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This is to certify that the
dissertation entitled

INVESTIGATION OF BILE STRESS TOLERANCE AND
IMMUNOMODULATORY PROPERTIES OF PROBIOTIC
LACTOBACILLUS REUTERI

presented by

KRISTI JAMES WHITEHEAD

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Microbiology and Molecular
Genetics

 

 

M 6:54.,

Major Professor’s Signature
“f /z=r /2 0o?
l I

Date

MSU is an Affirmative Action/Equal Opportunity Employer

PLACE IN RETURN BOX to remove this checkout from your record.
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5/08 K‘IProlecc&Pres/ClRC/DateDue.indd

 

INVESTIGATION OF BILE STRESS TOLERANCE AND
IMMUNOMODULATORY PROPERTIES OF PROBIOTIC LACTOBACILLUS
REUTERI
By

Kristi James Whitehead

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Microbiology & Molecular Genetics

2009

ABSTRACT
INVESTIGATION OF BILE STRESS TOLERANCE AND
IMMUNOMODULATORY PROPERTIES OF PROBIOTIC LACTOBACILLUS
REUTERI
BY
Kristi James Whitehead

This thesis focuses on characterization of different aspects Of L. reuteri
physiology that may contribute to the probiotic potential of the species. This
research includes an investigation of the bile stress response of L. reuteri ATCC
55730 (Chapter 2), analysis of the bile salt hydrolase activity of L. reuteri ATCC
PTA 6475 (Chapter 3), and the investigation of the potential immunomodulatory
role of lactobacillic acid, a cyclopropyl fatty acid that is specific to certain strains
of L. reuteri at particular stages of growth (Chapter 4).

The research contained within Chapter 2 includes an investigation of the
physiological response of L. reuteri ATCC 55730 to the bile stress that would be
encountered in the host small intestine, including an analysis of genes that are
important for survival and growth in the presence of bile. Three genes were
identified as being important for the survival of L. reuteri ATCC 55730 in bile: a
putative esterase, a clp chaperone, and a gene of unknown function that is
conserved within different L. reuteri isolates. One gene, encoding a multidrug
resistance protein in the major facilitator superfamily, was found to contribute to

the ability of the strain to grow in the presence of bile.

Chapter 3 examines the ability of L. reuteri ATCC PTA 6475 to
deconjugate bile acids. There is a strong correlation between strains isolated
from the gastrointestinal tract and the ability to deconjugate bile acids, thus it is
often suggested that bile salt hydrolase activity confers a selective advantage for
colonization of the gastrointestinal tract. L. reuteri ATCC 55730, the strain
investigated in the microarray and mutational analysis of the bile stress
response, exhibits a very low level of bile salt hydrolase activity. L. reuteri ATCC
PTA 6475, on the other hand, exhibits strong bile salt hydrolase activity against
all six major human bile acids. The research contained in Chapter 3 suggests
that this activity confers a growth advantage in the presence of bile acids at
slightly acidic pH levels.

Several strains of L. reuteri have been suggested to be able to modulate
the host immune response through the production of compounds that inhibit
production of the pro-inflammatory cytokine, TNF. The research in Chapter 4
identifies a possible novel immunomodulatory compound, lactobacillic acid,
which may be responsible for the observed suppression. Production of this
compound was demonstrated to be specific to L. reuteri strains with the TNF
suppressive activity, and importantly, the appearance of lactobacillic acid in cells
isolated from early stationary and late stationary phase cultures correlates with
the growth stages where the immunomodulatory activity is observed. Current
research isfocused on identifying the role of lactobacillic acid in TNF

suppression.

 

This work is dedicated to Daniel, who loves me unconditionally, makes me laugh,

and believes in me even when I don ’t believe in myself.

 

ACKNOWLEDGEMENTS

I would first and foremost like to acknowledge my advisor, Dr. Robert
Britton. I cannot thank you enough for the opportunity to work in your lab. You
have been a wonderful advisor, and you have played such a huge role in my
development as a scientist. Thank you so much for the patience that you have
demonstrated in getting me to think for myself and in helping me to become a
better public speaker. I am not sure that words can express how much the time I
have spent in your lab has meant to me. I truly believe that the day that I
decided to ask to join your lab was the day that I set myself on the path to
becoming a successful scientist. I hope to be able to continue using the skills
and lessons that l have learned from you as I continue on my scientific career.
Thank you. '

The members of my advisory committee have also been an invaluable
asset during my progress through graduate school. Dr. Cindy Arvidson, Dr. Lee
Kroos, Dr. Thomas Schmidt, Dr. Zeynep Ustunol, and Dr. Vincent Young have all
been amazing sources of knowledge and helpful discussions. Thank you all so
much for your patience and guidance.

To all of the past and present members of the Britton laboratory, thank you
so much for making the lab a great place to work. I thoroughly enjoyed all of the
debates and discussions, both scientific and non-scientific. Thank you all so
much for the daily support, as well as for putting up with me during the stressful

times! In particular, I would like to thank Dr. Laura Schaefer. I thoroughly

 

enjoyed getting to know you and spend time with you in the lab and outside of
the lab during my six years at Michigan State. You are a wonderful lab manager
and a great friend.

I would also like to acknowledge our collaborators: Jim Versalovic at
Baylor College of Medicine, Stefan Roos at the Swedish University of Agricultural
Sciences, and Eamonn Connolly at Biogaia. Thank you so much for lending your
expertise in the field of probiotic research. You each were a wonderful source of
information and guidance for many different aspects of my research. I also am
indebted to Dr. Babak Borhan in the Department of Chemistry at Michigan State
University and the members of his lab. This amazing group of scientists
welcomed me as one of their own, both in the laboratory and during lab
functions.

During my time at Michigan State, I developed friendships with some truly
wonderful individuals: Stephanie Eichorst, Kristin Huizinga, Jennifer Gray, Zarraz
Lee, Thu Nguyen, and Adam Mosey. Without each of you to talk to, to vent to
(and with), and to laugh with, I am not sure this journey would have been
possible; it certainly would have been much less enjoyable. I truly hope that we
can stay in contact with one another no matter where life takes us.

In addition, none of this would have happened if not for the love and
support that I have received both from my family and Daniel’s family. All of you
have played such a strong role in the development of who I am. Thank you so
much for supporting me through graduate school, especially after Daniel and I

announced that we would be moving far away to Michigan. We are headed back

vi

closer to home now, and we are looking fonlvard to spending more time with all of
you.

Finally, I am forever grateful for my husband Daniel. I am not sure I
believed you eleven years ago when you told me that we were meant to be
together after only a few dates, but you were so confident that I decided it might
be worth finding out where life took us. I could not be happier that you were
right. Through high school, college, and now graduate school, you have been
the absolute best friend and husband that anyone could ever ask for. You have
been a constant source of love and support, always believing that I could do
anything that I set my mind to. I am so proud of you and all that you have
accomplished as we have progressed through the last six years of graduate

school together.

vii

 

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. x

LIST OF FIGURES ............................................................................. xii

CHAPTER 1: THE PROBIOTIC POTENTIAL OF LACTOBACILLUS REUTERI

INTRODUCTION .......................................................................... 1
Prevalence .................................................................................. 2
Factors that aid in colonization ......................................................... 3
Proposed mechanisms of action ..................................................... 14
Animal studies and clinical trials ..................................................... 26
REFERENCES ........................................................................... 35

CHAPTER 2: GENOMIC AND GENETIC CHARACTERIZATION OF THE BILE
STRESS RESPONSE OF PROBIOTIC LACTOBACILLUS REUTERI ATCC
55730

INTRODUCTION ........................................................................ 44
MATERIALS AND METHODS ....................................................... 48
RESULTS ................................................................................. 54
DISCUSSION ............................................................................. 65
SUPPLEMENTAL INFORMATION .................................................. 72
ACKNOWLEDGEMENTS ............................................................. 82
REFERENCES ........................................................................... 83

CHAPTER 3: CHARACTERIZATION OF THE BILE SALT HYDROLASE
ACTIVITY OF LACTOBA CILLUS REUTERI ATCC PTA 6475

INTRODUCTION ........................................................................ 88
MATERIALS AND METHODS ....................................................... 91
RESULTS ................................................................................. 97
DISCUSSION ........................................................................... 109
ACKNOWLEDGEMENTS ............................................................ 115
REFERENCES ......................................................................... 116

CHAPTER . 4: IDENTIFICATION OF A PUTATIVE NOVEL
IMMUNOMODULATORY COMPOUND FROM LACTOBACILLUS REUTERI

INTRODUCTION ....................................................................... 120
MATERIALS AND METHODS ...................................................... 123
RESULTS ................................................................................ 129
DISCUSSION ........................................................................... 145

viii

ACKNOWLEDGEMENTS ............................................................ 148

REFERENCES ......................................................................... 149
CHAPTER 5: SUMMARY AND SIGNIFICANCE ...................................... 152
REFERENCES ........................................................................ 157

APPENDIX A: MICROARRAY ANALYSIS OF LACTOBACILLUS REUTERI
ATCC 55730 DURING MID-LOG AND EARLY STATIONARY PHASES OF

GROWTH
PURPOSE ............................................................................... 159
MATERIALS AND METHODS ...................................................... 159
RESULTS ................................................................................ 161
REFERENCES ......................................................................... 171

APPENDIX B: IDENTIFICATION OF A PUTATIVE BILE-INDUCIBLE
TRANSCRIPTIONAL ELEMENT FROM LACTOBACILLUS REUTERI

PURPOSE ............................................................................... 172
RESULTS ................................................................................ 173
FUTURE DIRECTIONS .............................................................. 175
REFERENCES ......................................................................... 176

APPENDIX C: INVESTIGATION OF A PUTATIVE MATRIX
METALLOPROTEASE FROM LACTOBACILLUS REUTERI

PURPOSE ............................................................................... 177
BIOINFORMATICS .................................................................... 178
EXPERIMENTS AND RESULTS ................................................... 180
DISCUSSION ........................................................................... 184
PROTOCOLS OF INTEREST ...................................................... 187
REFERENCES ......................................................................... 196

LIST OF TABLES

Table 2.1. Bacterial strains and plasmids used for this study ......................... 49
Table 2.2. Classes of genes differentially expressed during the first 15 minutes
of exposure to 0.5% bile ........................................................................ 57

Table 2.3. Classes of genes differentially expressed during growth in the
presence of 0.5% bile ........................................................................... 59

Table 2.4. Fold gene expression changes in the presence of bile for genes
chosen for disruption ............................................................................ 61

Table 2.5. Effects of 0.3% bile exposure on cell viability and final culture

density .............................................................................................. 62
Table 2.6. Genes over-expressed or under-expressed during both 15 minutes of
bile exposure (0.5% oxgall) and 15 minutes of acid stress (pH 2.7) ................ 69
Table 2.7. Genes over-expressed after 15 minutes of exposure to 0.5%

oxgall ................................................................................................ 72
Table 2.8. Genes over-expressed during growth in the presence of 0.5%

oxgall ................................................................................................ 75
Table 2.9. Genes under-expressed after 15 minutes of exposure to 0.5%

oxgall ................................................................................................ 76
Table 2.10. Genes under-expressed during growth in the presence of 0.5%
oxgall ................................................................................................ 79
Table 3.1. Bacterial strains and plasmids used for this study ........................ 92

Table 3.2. Doubling times and culture densities for L. reuteri ATCC PTA 6475
and PRBZ40 in the presence and absence of human bile acids ................... 104

Table 3.3. Observed growth effects of individual bile acids in plates or in liquid
medium on L. reuteri ATCC PTA 6475, PR8240, and L. reuteri ATCC 55730.
Notable differences between ATCC PTA 6475 and PRBZ40 are highlighted in
bold. ND = not determined. *Concentrations of greater than 0.1% were not
tested for the deconjugated bile acids due to solubility problems .................. 107

Table 4.1. Bacterial strains and plasmids used in this study ........................ 124

Table A1. Genes significantly over-expressed by L. reuteri ATCC 55730 during

 

early stationary phase ......................................................................... 163
Table A2. Genes significantly under-expressed by L. reuteri ATCC 55730 during
early stationary phase ......................................................................... 167
I
i
xi

LIST OF FIGURES

Figure 2.1. Representative growth curve of L. reuteri ATCC 55730 used for
microarray experiments (arrows represent time points where samples were taken
for RNA isolation). Open arrows represent samples for bile shock experiments;
filled arrows represent samples for bile adaptation experiments. 0.5% oxgall was
added at 250 minutes ........................................................................... 52

Figure 2.2. Comparison of survival after 30 minutes of exposure to 0.3% oxgall
for L. reuteri ATCC 55730 wild-type and PRB190 (lr1864 - CIpL), PRB188 (lr1516
- putative esterase), PRB167 (er085 - unknown), PRB114 (Ir1706 - Dps),
PRB126 (Ir1265 - multidrug resistance protein in the ABC transporter family), and
PRB130 (Ir1584 - multidrug resistance protein in the major facilitator superfamily)
mutant strains. Cultures were plated onto MRS plates after bile exposure to
determine the number of viable cells. Error bars represent standard deviation.
*p < 0.001 compared with wild-type ......................................................... 63

Figure 3.1. Different phenotypes observed using the plate test to determine bile
salt hydrolase activity. Strains of L. reuteri were plated onto MRS plates with or
without bile acids to determine bile salt hydrolase ability. A. L. reuteri ATCC PTA
6475 and the bile salt hydrolase mutant strain, PRBZ40, plated onto MRS plates
containing 0.5% oxgall. Precipitation of bile is observed from ATCC PTA 6475
as halos around the colonies; no activity is observed for PR8240. This
phenotype was also observed for plates containing glycodeoxycholate (GDCA)
and glycochenodeoxycholate (GCDCA) (data not shown). B. The two strains
(ATCC PTA 6475 and PR8240) plated onto MRS plates containing 0.01%
taurochenodeoxycholate (TCDCA). Bile salt hydrolase activity for this bile acid
was demonstrated by a cloudiness surrounding the colonies that could be
observed when the plates were held against a light. No activity is observed for
PR8240. C. ATCC PTA 6475 plated onto MRS or MRS plus 0.5%
taurodeoxycholate (TDCA). Deconjugation of TDCA was demonstrated by a
matte opaque granular appearance of the colonies. This phenotype was not
observed for PR8240 (data not shown) .................................................... 99

Figure 3.2. Deconjugated bile acids begin to accumulate after 4 hours in a
culture of L. reuteri ATCC PTA 6475. Representative TLC plate showing the bile
salt hydrolase activity of L. reuten' ATCC PTA 6475 against a synthetic “human”
bile acid mixture (SHB). Samples were taken a 1, 4, 7, and 23 hours. Control
solutions of cholate (CA) and deoxycholate (DCA) are included for comparison
[deoxycholate and chenodeoxycholate (CDCA) migrate at the same rate in this
solvent system]. The bile salt hydrolase mutant strain, PRB240, is shown to
have no activity at the 23-hour time point ................................................. 103

xii

Figure 3.3. Controlling the pH of the culture does not affect the bile salt
hydrolase activity of L. reuteri ATCC PTA 6475. Representative TLC plate
showing the bile salt hydrolase activity of L. reuteri ATCC PTA 6475 against a
synthetic “human” bile acid mixture (SHB) in MRS and buffered MRS. Samples
were taken at 7 hours. Control solutions of cholate (CA) and deoxycholate
(DCA) are included for comparison [deoxycholate and chenodeoxycholate
(CDCA) migrate at the same rate in this solvent system]. No difference in activity
is observed between cultures grown in MRS and cultures grown in buffered

MRS ................................................................................................ 106

Figure 4.1. Synthesis of cyclopropane fatty acids. Cyclopropane fatty acid
synthase converts oleic acid and vaccenic acid into dihydrosterculic acid and
lactobacillic acid, respectively ............................................................... 122

Figure 4.2. A representative growth curve for L. reuteri ATCC PTA 6475.
Samples for fatty acid analysis (mid-log, early stationary, and late stationary
phase) were collected at the time points represented by open circles ............ 127

Figure 4.3 - TNF-inhibitory strains produce lactobacillic acid. Cultures of five
strains of L. reuten' were grown in MRS broth for 18 hours at 37° C. Fatty acid
profiles were determined by gas chromatography. ATCC 55730 and CF48-3A
(TNF non-inhibitory strains) do not produce the CFA, lactobacillic acid. In
contrast TNF-inhibitory strains (ATCC PTA 6475, ATCC PTA 5289, and ATCC
PTA 4659) produce lactobacillic acid. Experiments were performed in triplicate;
error bars represent standard deviation. *Lactobacillic

acid ............................................................... 132

Figure 4.4. Lactobacillic acid appears in late stationary phase cultures of L.
reuten' ATCC PTA 6475. Cultures of L. reuteri ATCC PTA 6475 were grown in
MRS broth at 37°C. Samples were collected for FAME analysis at different
stages of growth. Experiments were performed in triplicate; error bars represent
standard deviation. *Lactobacillic acid ........................ 134

Figure 4.5. Disruption of NT01LR1143 (CFA synthase) eliminates production of
lactobacillic acid by L. reuteri ATCC PTA 6475. L. reuteri ATCC PTA 6475 and
PRB173 were grown overnight in MRS broth at 37° C. Samples were collected
for fatty acid analysis. Experiments were performed in triplicate; error bars
represent standard deviation ................................................................. 136

Figure 4.6. Lactobacillic acid production by L. reuteri ATCC PTA 6475 can be
increased by removal of Tween 80 from the growth medium. Overnight cultures
of L. reuteri ATCC PTA 6475 were grown in MRS A (complete MRS broth), MRS
B (MRS broth with Tween 80 and beef extract removed), or MRS C (MRS B plus
0.01 % cis-vaccenic acid); samples were collected for fatty acid analysis.

xiii

Experiments were performed in triplicate; error bars represent standard
deviation ........................................................................................... 139

Figure 4.7. An insertion in the CFA synthase gene of L. reuteri ATCC 55730 and
CF48-3A may prevent production of lactobacillic acid. PCR analysis revealed an
insertion in the region of the genome containing the cfa gene for both
immunomodulatory strains. This insertion results in the truncation of the cfa
gene. Small arrows represent the primers used for amplification of the region
surrounding the cfa gene ..................................................................... 141

Figure 4.8 - L. reuteri deficient in Cfa had diminished ability to suppress TNF.
Cell-free supernatants from L. reuten' cultured as planktonic cells or biofilms were
tested for the ability to inhibit LPS-activated THP-1 cells from producing TNF.
When cultured as planktonic cells (A) or biofilms (B), PBR173, which does not
produce lactobacillic acid, had diminished ability to suppress TNF production
compared to wild-type cells .................................................................. 144

Figure A.1. A representative growth curve of L. reuteri ATCC 55730. Samples
for microarray analysis (mid-log and early stationary phase) were taken at the
time point represented by the open circles ............................................... 162

Figure 8.1. The sequences of the 13 occurrences of the possible bile-inducible
transcriptional element in L. reuteri ATCC 55730 identified by MEME ............ 174

Figure C.1. The protein sequence of L. reuteri ATCC PTA 6475 NT01LR0595
(Lr1291 homolog). The putative cleavage site is marked with two forward
slashes (ll); this site was predicted by SignalP 3.0. The putative active site is
underlined, with the conserved residues in bold. The conserved methionine
residue (“met-turn”) is also in bold .......................................................... 179

xiv

 

CHAPTER 1

THE PROBIOTIC POTENTIAL OF LACTOBACILLUS REUTERI

Introduction

and. '

Probiotics, which are defined as “live microorganisms which when administered
in adequate amounts confer a health benefit on the host" (19), have become

increasingly popular over the past several decades as reports of antibiotic

 

resistant infections and the prevalence of gastrointestinal disorders such as
inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) continues
to rise. Despite the increase in usage, not much is known about the mechanisms
through which these microorganisms may be able to cause their beneficial
effects. Production of antimicrobial compounds, alterations of the host
microbiota, and modulation of the immune response of the host are among the
proposed mechanisms (18, 54, 62). In part because the mechanisms through
which these microbes cause their beneficial effects are not known, the
characteristics that make an effective probiotic are also speculative. Some
general characteristics that are proposed to be important include that the microbe
be of host origin, is nonpathogenic, is able to resist the stresses encountered
during passage through the host, and is able to confer a beneficial effect on the
host (18). Lactobacilli are among the most commonly used bacteria in probiotic

products, in part due to their long history of safe use in the food industry. Among

the lactobacilli, many different species are currently being investigated as
effective probiotics, including various strains of L. acidophilius, L. rhamnosus,
and L. reuteri (17). L. reuteri is a heterofermentive species of lactic acid bacteria

that is found natively in the gastrointestinal tracts of humans (52).

This review will summarize the recent literature regarding the potential of various
strains of L. reuteri to be used as effective probiotics; both in vitro experiments

and clinical trials will be discussed.

Prevalence

Although there is a lack Of knowledge regarding what characteristics make an
effective probiotic, it is generally considered to be important for the bacteria to
have been isolated from the host in which it will be used. Lactobacillus reuteri is
one of the few species of lactic acid bacteria that is naturally found in humans as
well as in a variety of animals as part of the normal microbiota. Using culture-
based isolation techniques, L. reuteri has been found to be one of the dominant
species of Lactobacillus isolated from chickens, mice, dogs, and pigs (2, 39, 41,
48, 79, 89). Within the human gastrointestinal tract, only a handful of lactic acid
bacteria are considered to be autochthonous; these include L. reuteri, L. gasseri,
and possibly L. ruminus and L. salivarius (52). Although bacterial samples are
often taken from the intestines or fecal samples for analysis, there are several

reports that suggest that L. reuteri may also inhabit the stomach of humans (52,

57, 78), a location typically considered to be fairly uninhabitable for bacteria due
to the extremely low pH. In a recent 28-day clinical trial, Valeur et al (78)
administered L. reuteri ATCC 55730, a probiotic strain of human-origin, to
participants. They were able to demonstrate that the strain survived in the
stomach, duodenum, and ileum of the study participants. This supports the
argument that strains isolated from the host are most likely already adapted for
survival in the host, and therefore, may be strains worth investigating for probiotic

usage.
Factors that aid in colonization.

Although being of host origin is generally considered to be an important trait,
many of the potential probiotic strains currently in use originated from the food
industry. As the definition of a probiotic requires the organism to survive
passage through the gastrointestinal tract, there are several characteristics that
are generally investigated through in vitro assays before the strains are used in
animal studies or clinical trials. These traits include resistance to stresses
encountered in the gastrointestinal tract (such as low pH in the stomach and bile
in the small intestine), as well as the ability to attach to host surfaces for
colonization. It is also generally desirable to understand how strains are able to
survive these harsh conditions. Knowledge of the mechanisms could allow
researchers to quickly screen for strains that can survive passage through the

gastrointestinal tract based on a particular trait or allow for improvement of the

survival rate of a strain with intriguing beneficial properties that may have poor
survival or colonization abilities. As multiple strains of L. reuteri have been
shown to survive in the gastrointestinal tract in a wide variety of animals, this

species may be ideal for investigating these mechanisms.

Acid stress. As previously mentioned, several studies have shown that L.
reuten' can survive in the human stomach (52, 57, 78), an environment generally
considered to be inhospitable to bacteria due to its extremely low pH. The pH in
the human stomach can reach as low as 1.5 during the fasting stage, with values
ranging between 3.0 to 5.0 after consumption of a meal (16). One of the most
commonly used delivery mechanisms for probiotics, yogurt and other fermented
dairy products, are also typically acidic (16). Therefore, an understanding of the
ways in which various strains of L. reuteri can survive lOw pH exposure is

beneficial for host, as well as industrial, applications.

Lee et al (37) exposed L. reuteri ATCC 23272 to pHs of 5.0, 4.5, and 4.0 for one
hour and compared the protein expression under these conditions to cultures
grown at pH 6.8. They identified 40 proteins involved in a wide range of cellular
functions that were significantly differentially expressed during incubation at all
three lower pH values, suggesting that L. reuteri has a complex response to pH
stress. Proteins over-expressed during growth at lower pHs included those
involVed in transport and binding, transcription and translation, metabolism,

stress, and pH homeostasis.

 

In a more focused study, microarray and mutational analyses of the acid shock
response of L. reuteri ATCC 55730 were performed by Wall et al (81 ). The gene
expression changes of cells that were exposed to pH 2.7 for either 5 or 15
minutes were identified based on comparison with the gene expression profiles
of cells grown at pH 5.1. Overall, 72 genes were found to be differentially
expressed when L. reuteri ATCC 55730 was exposed to acid shock conditions.
Viability studies at pH 2.7 revealed that for this strain, more than 80% of the cells
survived exposure for one hour, although no growth appeared to take place.
This suggests that although the strain is able to survive passage through the
stomach and even possibly colonize the stomach (78), time may be needed to
adapt to the harsh acidic conditions before growth can resume. Such an
adaptation period lasting approximately two hours was demonstrated when L.
reuteri ATCC 55730 was exposed to bile in vitro (87). Based on the microarray
analysis, two genes were chosen for disruption; lr1864 (clpL, a putative ATPase
that may act as a chaperone) and lr1516 (a putative esterase in the penicillin
binding protein family). Both of these mutants were found to have significantly
lower survival rates compared to the wild-type strain when incubated in a
synthetic gastric juice at pH 2.0 for 50 minutes. The mutational analysis
suggests that the CIpL chaperone may be needed to aid the cells in degrading or
refolding proteins denatured by the low pH exposure; it also suggests that the
putative esterase may be involved in altering the cell wall in such a way that

allows the cells to survive exposure to low pH (81). Further study is needed to

fully understand how L. reuteri responds to and survives exposure to the low pH
found in the stomach. The importance of CIpL and the putative esterase should
be investigated in an in vivo model to determine if they truly contribute to survival

in the gastrointestinal tract.

Bile stress. After exposure to the low pH in the stomach, probiotic bacteria then
move into the small intestine where they are exposed to bile, a complex mixture
that typically aids in fat digestion and absorption (27). Bile acids, the main
component in bile, are known to have potent antimicrobial effects (25), yet as
previously mentioned, some strains of L. reuteri have been shown to colonize
portions of the small intestine (78). The response of these strains to bile is
important for understanding what physiological changes the bacteria undergo

during passage through the gastrointestinal tract.

Bile acids are amphipathic molecules that function to solubilize fats. Their
amphipathic nature allows them to form mixed micelles with fats and cholesterol
in the body and also allows them to act as detergents. One of the main ways
through which bile is thought tO cause damage to bacterial cells is through
disruption of the cell membrane and wall. Taranto et al (71) demonstrated that
growth in the presence of bile induced changes in the cell membrane of L. reuteri
CRL 1098. Exposure to bile decreased the amount of phospholipids and also

decreased the ratio of saturated to unsaturated fatty acids in the cell membrane.

These changes may be important for survival during passage through the

gastrointestinal tract.

L. reuteri ATCC 55730 was shown to survive in physiologically relevant
concentrations of bile in vitro (87). Growth studies revealed that there was an
initial pause in growth when mid-log phase cells were exposed to bile. This
pause in growth is believed to be a period of adaptation, during which the cells
make physiological changes that allow them to then grow in the presence of bile.
In order to determine how bile exposure affects the gene expression of L. reuteri
and to understand what changes allow the cells to resume growth in the
presence of bile, microarray analyses were performed comparing the gene
expression profiles of cells before and after bile exposure. Based on the
microarray analysis, several genes were chosen for mutational analysis.
Disruptions in three genes were shown to cause significantly lower survival rates
during the first 30 minutes of bile exposure. These genes included lr1864 (clpL),
lr0085 (a gene of unknown function that is conserved among L. reuteri only), and
lr1516 (a putative esterase). In addition, a disruption in another gene, lr1584 (a
multidrug resistance protein in the major facilitator superfamily), appeared to
affect the ability of the cells to grow in the presence of bile, although the initial
survival rate was not altered (87). The growth defect observed in the lr1584
mutant is in accordance with known mechanisms of bile resistance for gram-
negative organisms, where the utilization of efflux pumps to expel bile acids has

been demonstrated (25).

Interestingly, there is overlap in the gene expression changes of L. reuteri
exposed to low pH and bile based on microarray analysis (81, 87). There is also
overlap in the mutational analysis; ClpL (lr1864) and the putative esterase
(lr1516) were both shown to be important for survival in low pH and bile. This

overlap suggests that exposure to acid in the stomach may prepare L. reuteri for

Anna--1."

exposure to bile in the small intestine. Further research is required to determine
if the period of adaptation observed in vitro during bile exposure may be

decreased by a prior exposure to low pH.

 

One other possibility of how L. reuteri may deal with the stress of bile acids is
through the activity of a bile salt hydrolase (BSH) enzyme. When bile is secreted
into the small intestine from the gall bladder, the majority of bile acids are
conjugated to either a glycine or taurine molecule; however, by the time the bile
reaches the end of the small intestine, almost all of the bile acids are
unconjugated (i.e. the glycine or taurine has been removed) (47). This
deconjugation activity is due to bacterial bile salt hydrolase enzymes and plays a
role in bile acid metabolism of the host (7). There is much debate as to the role
of the bacterial BSH enzymes. Some possible roles include lowering the
cholesterol of the host, providing a nutritional benefit to the bacteria, or
detoxification of the bile acids to protect the bacteria (7). Taranto et al (74)
demonstrated that L. reuteri CRL 1098 contained an intracellular bile salt

hydrolase enzyme that was expressed when the cells were in stationary phase.

Whether or not this enzyme plays a role in bile resistance of L. reuteri is yet to be
determined. Research in our lab suggests that there is substantial strain
variation in BSH enzyme specificity and activity among various L. reuteri

(unpublished observation).

Attachment. It has been suggested that probiotic bacteria should persist in the
gastrointestinal tract in order to cause their beneficial effects; the ability to attach
to intestinal surfaces such as epithelial cells or mucus is often proposed to aid in
this persistence. Several studies have examined the ability of L. reuteri to
adhere to mucosal surfaces using cell lines, mucus components, or animal

models.

Wang et al (85) tested nine different strains of Lactobacillus for their ability to
attach to human enterocyte-like HT-29 cells, as well as to porcine gastric mucin,
one of the main components of intestinal mucus. Of the nine strains, they found
that L. reuteri JCM1081, a chicken isolate, was best able to adhere. Further
analysis identified a 29 kDa protein, a putative ATP-binding cassette transporter
protein, that was able to bind to both the cell line and the mucin in vitro,
suggesting that this protein may be responsible for the adherence observed. The
authors propose that based on these experiments mucin may actually act as a
receptor for the binding of probiotic bacteria in the gastrointestinal tract. Roos et
al (55) also previously identified a 29 kDa collagen binding protein from L. reuteri

NCIP 11951 similar to ABC-type transport proteins that was suggested to aid in

attachment of the bacterium to epithelial surfaces. Sequence comparison
revealed these two proteins to be similar (85), suggesting a conserved binding

protein between the two strains.

Although there may be conserved traits identified, strain specificity is something
that should be taken into careful consideration when investigating L. reuteri for
probiotic usage. Characteristics that may be important for causing beneficial
effects, such as binding to intestinal surfaces, may vary substantially between
strains. For example, Miyoshi et al (42) tested L. reuteri strains from various
sources (human, pig, mouse, and chicken) for their ability to adhere to Caco-2
cells in vitro. They identified three strains that had high levels of adherence (L.
reuteri DSMZ 20016, 104R, and LEM83) and two strains that had low levels of
adherence (L. reuteri ML1 and LB54). Upon further investigation, they identified
a protein, MapA (mucus adhesion promoting protein) that also bound to Caco-2
cells and had the ability to inhibit the binding of L. reuteri to Caco-2 cells in a
concentration-dependent manner. Although MapA appears to be a protein that is
involved in binding to eukaryotic cells, all strains utilized in this study were found
to contain the gene for MapA, despite the different adherence levels. Also,
although MapA was able to compete with L. reuteri for binding, approximately
50% of the bacterial cells were able to bind to Caco—2 cells, even under
conditions using saturating amounts of MapA. These observations suggest that
binding to Caco-2 cells involves more than one factor, and that binding activity

most likely is controlled by different factors in different strains of L. reuteri.

10

Although some studies have investigated binding to both cells and mucus or
mucus components, other studies have focused singularly on the ability of L.
reuteri to bind to mucus, since the epithelial surface in the gastrointestinal tract is
covered with a mucus layer in healthy individuals. Jonsson et al (31)
investigated the ability of seven different strains of L. reuteri to bind to pig gastric
mucin. They observed varying levels of attachment between the strains. When
the strains were grown in the presence of mucin, an increase in adherence was
observed for strains with previously low levels of binding, whereas an inhibition of
binding was observed for strains with naturally high levels of adherence.
Proteinase K treatment was found to abolish binding, suggesting that binding is
due to a protein. The effects observed by growth of the strains in the presence of
mucin could also be explained by the dependence of binding on a protein or
proteins. Growth in mucin could induce expression of a protein necessary for
binding in the strains with naturally low levels of binding, but the presence of
mucin could compete with a binding protein already present in the strains with
naturally high levels of binding. This binding is not believed to be due to the
previously identified 358 kDa cell surface protein, Mub, which was identified from
L. reuteri 1063 and shown to bind to mucus and mucus components in vitro (56),
as only one of the seven strains included in this study is known to produce Mub

(31).

11

Although in vitro adherence tests provide a rapid means of examining the
potential of certain strains to persist in the intestine, little is known about how
host factors and physiological changes that the bacteria undergo during passage
through the host may affect persistence. Initial studies in the host have identified
a wide range of factors that can affect colonization success, including various cell
surface proteins, exopolysaccharide production, and D-alanylation of teichoic

acids in the cell membrane and wall.

Walter et al (82) examined the effect of disruption of four genes on the survival of
L. reuteri 100-23 in the mouse. They identified one mutant with a disruption in
Lsp (a high-molecular—mass surface protein) that was delayed in its colonization
rate and which had impaired adherence to the forestomach epithelium of the
mouse. This mutant was also found exhibit lower colonization levels when
inoculated in competition with the wild-type strain. These experiments suggest
that cell surface proteins are important for adherence and colonization in vivo,
supporting the previously described in vitro data. The study also demonstrated
somewhat reduced performance in an MsrB mutant (methionine sulfoxide

reductase), although there was large inter-animal variability for this mutant.

Exopolysaccharide (EPS) production may also play a role in persistence for
some strains. In L. reuteri TMW1.106, two proteins involved in EPS production
were found to affect colonization of the strain in the gastrointestinal tract of mice.

thA (glucosyltranferase) and lnu (inulosucrase) mutants were both found to be

12

defective in colonization of the mouse. The inu mutant was defective in
colonization when put into competition with the wild-type strain; the 9th mutant
was defective in colonization when it was inoculated in competition with L.
johnsonii #21, but not when it was inoculated in competition with the wild-type L.
reuteri, suggesting that the wild-type strain was somehow able to complement

the observed defect in the mutant (84).

Finally, the effect of D-alanylation of teichoic acids in the bacterial cell membrane
and wall has been investigated in L. reuteri. The incorporation of D-alanyl esters
into teichoic acids has been shown to be important for bacterial virulence when
studied in various pathogens (1, 15, 51). In order to investigate whether there is
overlap between the methods that pathogens and beneficial bacteria use to
colonize the host, Walter et al (83) disrupted the D-alanylation operon of L.
reuten' 100-23 by insertional disruption of dltA, the second gene in this operon.
The dltA mutant was defective for colonization of the mouse when inoculated in
competition with the wild-type strain, as well as when it was inoculated alone.
The mutant also was demonstrated to be defective in the ability to form a biofilm
on the murine forestomach epithelium. In vitro studies shoWed that the mutant
had impaired growth under acidic conditions, which may play a role in the

observed in vivo defects.

Overall, attachment to mucosal surfaces is a complex phenomenon that appears

to be dependent on multiple bacterial surface characteristics such as EPS

13

production and cell surface proteins. Although there may be some conservation
of characteristics important for attachment and persistence, there also appears to
be considerable strain variation with L. reuteri that should be taken into

consideration.

Proposed Mechanisms of Action

Although the popularity of probiotics has increased over the last few decades and
they are being investigated as possible treatments for a wide variety of ailments,
the mechanisms of action for the beneficial effects observed with probiotic
treatment are still unknown. Some of the proposed mechanisms of action
include pathogen inhibition, primarily through the production of antimicrobial
compounds, and modulation of the host immune response (54, 62). Various

strains of L. reuteri have been investigated for these traits.

Pathogen inhibition - production of antimicrobial compounds. Although
production of hydrogen peroxide and lactic acid are commonly thought to
contribute to pathogen inhibition by lactic acid bacteria (34, 35), most of the
research regarding pathogen inhibition by L. reuteri has focused on other
antimicrobial factors. L. reuteri is known to produce at least three other separate
antimicrobial compounds: reuterin, a by—product of glycerol metabolism,

reutericylcin, a novel tetramic acid derivative, and reutericin, a bacteriocin.

14

Reuterin. One of the most widely studied antimicrobial compounds produced by
L. reuteri, and the reason commonly attributed for the beneficial effects observed
with administration of the species, is reuterin, a by-product of glycerol
metabolism. Reuterin has been identified as an equilibrium mixture of 3-
hydroxypropionaldehyde (3-HPA) in the monomer form, the hydrate form, and
the cyclical dimer form (70). Reuterin has been found to have a wide range of
antimicrobial activity, including antibacterial, antimycotic, and antiprotozoal
activities (3, 13). Lactobacilli are generally more resistant to reuterin than other
microbes; one study found that at least two times the reuterin was needed to kill
lactic acid bacteria as compared to other bacteria (13, 14). Although other
organisms have been shown to produce 3-HPA as a by-product of glycerol
metabolism (5, 20, 21, 40, 60), L. reuteri appears to be fairly unique in the ability
to accumulate large amounts of reuterin (although this trait is possibly shared
with certain strains of L. coryniformis (40)). A recent study demonstrated that L.
reuteri had the highest reuterin Mle (minimum inhibitory concentrations) when

compared to other intestinal bacteria (14).

Recent work has focused on the possible mechanism of action for reuterin’s
antimicrobial effects, as well as on attempts at determining whether reuterin is a
relevant antimicrobial compound in vivo. Morita et al (44) were able to show that
reuterin can be produced in detectable amounts in vivo using gnotobiotic BALB/c
mice monoassociated with L. reuteri JCM 1112 if glycerol is present. Their

research also demonstrated that 7 to 10 mM glycerol was found in the fecal

15

material of untreated mice, suggesting that reuterin may be produced in

concentrations relevant for antimicrobial activity in vivo.

Schaefer et al (63) have suggested that reuterin causes its broad-spectrum
antimicrobial effects by triggering oxidative stress through modification of thiol
groups. This mechanism is supported by an overlap in the gene expression
profile of Escherichia coli cells treated with reuterin and the known OxyR regulon,
a group of genes known to be involved in responding to various oxidative
stresses. The modification of thiol groups suggests that the aldehyde form of 3-

HPA is most likely the active portion of the equilibrium mixture (63).

One intriguing characteristic about reuterin production is that larger amounts of
reuterin are accumulated in the media when other bacteria are present. Various
bacteria have been shown to cause this increased accumulation of reuterin (13,
63). Schaefer et aI recently observed that live bacteria and UV-killed bacteria
but not heat—killed bacteria, triggered this increased accumulation (63). It has
not been determined whether the excess accumulation is due to increased

reuterin production or increased secretion of reuterin.

Although reuterin is commonly attributed as one of the main reasons for the
observed beneficial effects with L. reuteri, care should be taken to confirm this as
the active trait for individual strains and in specific instances. Despite the

evidence that reuterin is produced by various strains of L. reuteri, accumulation

16

levels can differ (69), and some strains have been shown to not contain the
necessary enzymes for reuterin production (8). Some research also suggests
that the genes necessary for reuterin production may have been horizontally
acquired (44), possibly explaining the lack of these enzymes in certain strains. L.
reuteri RC-14, a strain demonstrated to have many beneficial effects in urogenital
health, does not make reuterin and does not contain the glycerol dehydratase
enzyme necessary for converting glycerol into 3-HPA. Therefore, any probiotic
effects observed with this strain are not due to reuterin production (8), and other

possible mechanisms of action should be explored.

Reutericyclin. Reutericyclin was first identified as a low-molecular weight
antimicrobial compound that is produced by L. reuten' LTH2584, a strain isolated
from sourdough bread fermentations (22), although more recent research has
demonstrated that it is produced by several different strains isolated from
sourdough fermentations (23). Holtzel et al (29) identified reutericyclin as a novel
tetramic acid derivative, 3-acetyl-1-(2-trans-decenoyl)-2-hydroxy-(5R)-isobutyl-A2-
pyrroline-4-one. Reutericyclin has been shown to have a broad-spectrum
antimicrobial activity against various gram-positive organisms, including
Lactobacillus spp., Bacillus subtilis, Bacillus cereus, Enterococcus faecalis,
Staphylococcus aureus, and Listeria innocua (22). Ganzle et al (24)
demonstrated that the antimicrobial activity observed for reutericyclin was due to
its ability to dissipate the transmembrane proton potential of sensitive cells. The

main role of reutericyclin has been proposed as contributing to the persistence of

17

L. reuteri in sourdough fermentations. This proposed role is due to exhibited
production of active amounts of reutericyclin in sourdough fermentations by L.
reuteri (23), although due to the wide-spectrum inhibitory activity of the
compound against gram-positive bacteria, further investigation of this compound

is warranted.

Reutericin. Bacteriocins are antimicrobial proteinaceous compounds that have
activity against closely related bacteria (53). At least one strain of L. reuteri, L.
reuteri LA6, is known to produce a bacteriocin, reutericin 6. Reutericin 6 is a 2.7
kDa protein (32) that has been shown to have bacteriocidal activity against other
lactobacilli, including L. acidophilus and L. delbruckii. The role of reutericin 6 has
been proposed as a way for L. reuteri to remain the dominant species of
heterofermentive lactobacilli in the gut (75). Reutericin 6 and gassericin A (a
bacteriocin produced by Lactobacillus gasseri LA39) have been demonstrated to
be identical in their molecular weight and primary amino acid structure, yet vary
in the spectrum of their antimicrobial activity. The proposed reason for this
difference in activity is a difference in the D-amino acid content of the two
proteins (33). Investigation into the specificities of these two compounds is
needed to determine the role of these similar compounds that are both produced
by bacteria isolated from the same location, the feces of a human infant (33).
Further research into whether these two organisms use the compounds to
compete or to cooperate could indicate whether a mixture of these two strains

might be advantageous for probiotic usage.

18

Pathogen inhibition - displacement. Apart from actually inhibiting the growth
of or killing various pathogens, it is also possible that probiotics such as L. reuteri
exert their beneficial effects through competition for binding sites or nutrients.
Various studies have investigated the ability of L. reuteri to either inhibit the

attachment of pathogens or to displace pathogens that have already adhered.

Strain specificity appears to have a large effect on the ability of L. reuteri to inhibit
binding of pathogens to epithelial cells. Vesterlund et al (80) found that L. reuteri
ING1 not only had significantly lower adherence levels to mucus than
Staphylococcus aureus, but the strain also was not able to displace adherent S.
aureus, unlike other lactic acid bacteria tested in the study. They were, however,
able to demonstrate a significant reduction in the viability of S. aureus when
incubated with L. reuteri ING1 in the presence of 1% glycerol, suggesting that

this strain may still be effective in preventing infection by S. aureus.

It is also important to consider the effects that certain probiotic strains may have
on one another, since many probiotic companies are marketing products
containing mixtures of various strains. Larsen et al (35) tested multiple strains of
Lactobacillus, including three strains of L. reuteri for the ability to bind to porcine
epithelial cells and to either inhibit binding of or promote displacement of other
lactobacilli or pathogenic E. coli 0138 using in vitro assays. The authors of this

study not only demonstrated a wide range in the adherence capabilities of the

19

three L. reuteri strains (ranging from 3.5 to 38% of bacteria), but also found that
the most strongly adherent strain, L. reuteri DSM 12246, could actually displace
other lactobacilli. Interestingly, in this study, there appeared to be no correlation
between the ability of the lactobacilli to adhere and the inhibition of the E. coli

0138, as all tested lactobacilli reduced adherence of the E. coli (35).

One possibility for inhibition of binding or displacement of pathogens is through
competition for binding sites. Todoriki et al (76) tested various strains of
lactobacilli for the ability to adhere to Caco-2 cells in vitro. They demonstrated
that a strain of L. reuteri, JCM 1081, had the highest level of adhesion, while L.
crispatus JCM 8779 had the second highest level of adhesion. Both of these
strains were able to lower adherence levels Of E. coli, Salmonella typhimun'um,
and Enterococcus faecalis. Although the adherence inhibition was suggested to
be due to antimicrobial activity in the L. crispatus culture, no such activity was
observed with the L. reuteri culture, suggesting either competition for binding
sites or steric hindrance was the cause of the observed adherence inhibition.
Using a more specific example, out of nine L. reuteri strains tested, two were
shown to not only bind to both putative glycolipid receptor molecules of
Helicobacter pylori, but also to inhibit binding of H. pylori to both, suggesting a
competition for receptor binding (45). Another interesting connection regarding
competition for binding sites is that Heinemann et al (26) identified a 29 kDa
protein (as part of a biosurfactant mixture) from L. reuteri RC-14 that has strong

anti-adhesive properties against Enterococcus faecalis 1131. The authors

20

suggest that this protein is the same one identified by Roos et al (55) that is
believed to play a role in adherence of L. reuteri NCIB 11951. The identification
of the same protein during studies investigating adhesion of a probiotic strain to
host tissues and protection of host tissues by a probiotic strain suggest there
may be similarities in the adherence mechanisms of probiotic and pathogenic

strains which could lead to competition for binding sites.

One study has shown that L. reuteri may not only be able to displace individually
bound bacteria, but also can have an effect on bacterial communities such as
biofilms. L. reuteri RC-14, a strain demonstrated to have beneficial effects on
urogenital health, was tested to determine its effect on biofilms formed by
Gardnerella vagina/is, a pathogen commonly associated with bacterial vaginosis.
L. reuteri RC-14 was found to disrupt and invade G. vagina/is biofilms in vitro, as
well as cause a significant reduction in viability of the pathogen after co-
incubation for 24 hours (59), suggesting that this strain can have a detrimental

effect on an already well-established pathogen.

Pathogen inhibition - alteration of virulence factor expression. One other
possibility for probiotic benefit through direct inhibition of pathogens is the
possibility that the probiotic bacteria are able to alter either gene or protein
expression of the pathogen’s virulence factors. There are several in vitro studies
that suggest that this may be yet another possible mechanism of action for L.

reuteri.

21

Several studies have investigated the response of E. coli O157:H7 to incubation
with L. reuteri. Carey et al (11) demonstrated that various strains of
Bifidobacterium, Pediococcus, and Lactobacillus, including L. reuteri were able to
decrease expression of Stx2A, the gene that encodes the A subunit of Shiga
toxin 2. A different study demonstrated that culture supernatants from a certain
strain of L. reuteri, L. reuteri ATCC 55730, were able to repress E. coli O157:H7
ler expression. The main virulence factors for E. coli O157:H7 are found in a
pathogenicity island termed the locus of enterocyte effacement or LEE. The
regulator of expression for this pathogenicity island is ler (or LEE regulator). The
ability of L. reuteri culture supernatants to suppress expression of ler was found
to be strain dependent, as supernatants from L. reuteri ATCC 55730 repressed
expression, but supernatants from L. reuten' 100-23 actually induced expression

of Ier (30).

Protein expression from pathogens can also be affected, as evidenced by co-
incubation of Staphylococcus aureus with L. reuteri RC-14 (36). Research
demonstrated that secreted molecules of L. reuteri RC-14 were able to trigger a
decrease in expression of SSL11 (staphylococcal superantigen-Iike protein 11)
by S. aureus. SSL11 is a putative exotoxin that is proposed to be a virulence
factor for S. aureus. The authors also demonstrated that the secreted molecules
responsible for the suppression were not sensitive to protease treatment,

suggesting that proteins are not responsible for the observed effects. The

22

responsible secreted molecules were shown to repress the SSL11 and P3
promoters of S. aureus, further suggesting that L. reuteri RC-14 may be able to

alter the virulence of this common pathogen (36).

Immunomodulation. Apart from the potential for probiotic strains to cause their
beneficial effects by either altering the microbial community of the host through
production of antimicrobial compounds or through the prevention of
establishment of various pathogens, one other popular proposed mechanism of
action is the ability of probiotic bacteria to modulate the immune response of the
host. Various studies have been performed to investigate the effect that
administration of L. reuteri may have on dendritic cell maturation and cytokine

production.

Dendritic cells are antigen-presenting cells that have the capability to direct the
immune response of the host through the stimulation of B and T lymphocytes (4);
probiotic strains that have the capability to affect the maturation of dendritic cells
could therefore provide a potent modulation of the immune response.
Christensen et al (12) investigated the ability of six different strains of lactobacilli
to affect dendritic cell maturation by measuring cytokine production and surface
marker expression. They were able to demonstrate that the irradiated bacteria
had varying effects, with L. reuteri DSM 12246 exhibiting the weakest effect on
most surface markers and cytokines measured. L. casei CHCC 3139, on the

other hand, was shown to have the strongest effects on surface markers and IL-

23

12 of the strains measured. Interestingly, when L. reuteri was added along with
L. casei, L. reuteri was able to inhibit the increase in pro-inflammatory cytokines

lL-12, IL-6, TNF, and the surface marker CD86 observed with L. casei alone.

Several other studies have also investigated the response of dendritic cells to
certain strains of L. reuteri and found significant differences based on the strain
tested. Mohamadzadeh et al (43) determined that L. reuteri ATCC 23272 was
able to activate and induce maturation of dendritic cells and trigger an increase in
pro-inflammatory lL-12 production, suggesting that the L. reuteri strain was
shifting the dendritic cells towards polarization of a Th1 response. The authors
observed similar effects with both live and irradiated cells, suggesting that
bacterial viability was not essential for this effect. On the other hand, Smits et al
(67) found no effect on cytokine production by dendritic cells when they were co-
incubated with L. reuteri ASM 20016; however, they were able to determine that
the dendritic cells had been “primed” to drive the development of T regulatory
cells that then led to an increase in lL-10 production by T helper cells. lL-10 is
typically considered to be involved in anti-inflammatory responses. The authors
of this study also demonstrated differing effects based on the ratio of bacteria to
dendritic cells. They showed that a ratio of bacteria to dendritic cells that was
higher than 1:1 actually resulted in a decreased priming effect, suggesting that
care should be taken when choosing the dosage amount of probiotics. These
studies emphasize the need for thoroughly testing probiotic strains to determine

their immunomodulatory effects before using them individually or in combination

24

as treatments, as some strains of L. reuteri promoted production of lL-12 (pro-

inflammatory) and others promoted production of lL-10 (anti-inflammatory).

Several labs have investigated the effect of probiotic administration on the
production of the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF).
Soria et al (68) showed that L. reuteri CRL 1098 was able to decrease TNF
production, while L. acidophilus CRL 1014 and L. rhamnosus CRL 1036
increased production of TNF by human peripheral blood mononuclear cells,
demonstrating again that some probiotic strains may be considered
immunosuppressive, while others may be considered immunostimulatory. The
effects on TNF production were only observed with live bacterial cells, and these
effects were suggested to be somehow involved with lipid rafts. Lipid rafts are
microdomains found in eukaryotic plasma membranes that have been shown to
be involved in interactions with pathogenic bacteria. This study again suggests
an overlap in the mechanisms through which probiotics and pathogens interact
with eukaryotic cells, despite the fact that the outcomes of these interactions are

Often very different.

Although the previous study demonstrated a difference in the effects of probiotics
of different species of Lactobacillus, variation in the effect of different strains of L.
reuteri has also been observed. A study comparing three strains of L. reuten',
ATCC PTA 6475, ATCC 55730, and CF48-3A, found that only L. reuteri ATCC

PTA 6475 was able to suppress TNF production from LPS-activated monocytes

25

and macrophages (38). The suppression was shown to be due to secreted
compounds produced by the bacteria, and the effect was demonstrated for both
human cell lines and monocyte-derived macrophages isolated from Crohn's
disease patients. Lin et al (38) offer evidence that suggests that the TNF
suppression observed with cell-free supernatant from L. reuteri ATCC PTA 6475
may be caused by transcriptional regulation of TNF. The authors also note that
there were differences in the responses of the isolated macrophages from
different individuals, suggesting that the genetic background of the host may also
play an important role in the interaction. A particularly intriguing observation is
that cell-free conditioned medium from L. reuteri ATCC PTA 6475 was able to
suppress TNF to similar levels as the current Crohn’s disease treatment,
infiiximab (a TNF antibody), suggesting that probiotics may have potential as

treatments for IBD (38).

Animal studies and clinical trials.

The most common studies performed on potential probiotic strains are in vitro
tests to investigate whether the strains demonstrate the “necessary”
characteristics to provide probiotic benefit, including survival to stresses in the
gastrointestinal tract (acid and bile), the ability to attach to mucus or epithelial
surfaces, the ability to inhibit or displace pathogens, and the ability to modify the
host immune response (18, 54, 62). Although these characteristics are often

considered to be important for a bacterial strain to be able to cause beneficial

26

effects, the mechanism of action for most of the beneficial effects attributed to
probiotic strains is unknown, and therefore, what characteristics make a strain
“effective” are also unknown. For example, Van Coillie et al (79) carefully
screened strains of Lactobacillus isolated from hens for various characteristics
such as production of antimicrobial acids and adherence to epithelial cells.
Although they were able to identify a strain of L. reuteri that did decrease counts
of Salmonella enterica from inoculated chicks, they reached the conclusion that
the in vitro trait selection did not equal in vivo effectiveness. The authors suggest
that this difference may be due to host-associated factors that are not included
during in vitro testing (79). Therefore, the ultimate test for a potential probiotic
strain comes in the form of in vivo studies, including animal models of disease,
and ultimately, clinical trials. There are many animal studies and clinical trials

involving L. reuteri; a selected few will be discussed here.

Diarrhea. L. reuteri has been shown to be effective against diarrhea of various
causes in children. Shornikova et al (65, 66) demonstrated that L. reuteri could
actually be used as a treatment to reduce the duration of diarrhea (primarily from
rotavirus) in children. In this study, the probiotic was administered after children
were brought to the hospital for treatment for the diarrhea. In another study, it
was found that children in a daycare that received L. reuteri ATCC 55730 had
significantly less diarrhea episodes and the duration of these episodes was

shorter than in the control group Of children (86).

27

Inflammatory bowel disease (IBD). Inflammatory bowel disease is a set of
gastrointestinal disorders marked by chronic inflammation that affects a smaller
portion of the population and is more severe than lBS. IBD can be divided into
Crohn’s disease or ulcerative colitis, depending on the prevalence and location of
inflammation. The prevalence of IBD has increased over the last few decades,
particularly in regions with historically low occurrence (88). The proposed causes
of IBD include: an overly aggressive immune response to commensal bacteria
due to genetic factors or irregular immune response of the host, the presence or
absence of particular bacteria, or environmental factors (58, 64). Probiotics,
which are proposed to act through alteration of host immune responses or
alteration of host microbial communities, are proposed as possible therapies for

IBD (58, 64).

Several studies have shown that lL-10 deficient mice, which are prone to
spontaneous colitis unless raised in germ-free conditions, have a difference in
their lactic acid bacteria population than wild-type control mice. Both Madsen
(39) and Pena (48) found that the dominant Lactobacillus in wild-type control
mice was L. reuteri, while the dominant type in lL-10 deficient mice was L.
johnsonii. These studies not only suggest again that host genetic background
can affect the microbial community, but also suggest that the presence of certain
lactobacilli, including L. reuteri, may prevent spontaneous colitis. In fact, Madsen
(39) was able to show that by repopulating lL-10 deficient mice with L. reuteri, the

development of colitis was prevented, although the study also showed that

28

increasing the amount of lactic acid bacteria in the gut by lactulose treatment
also prevented colitis, so the effect of L. reuteri therapy observed in that

particular study may not be specific to L. reuteri.

Probiotic L. reuteri has also been demonstrated to have some level of
effectiveness against various rodent models of induced colitis, including
Helicobacter hepaticus-induced (49), acetic acid-induced (28), and TNBS
(trinitrobenzenesulfonic acid)-induced (50). Pena (49) showed that inflammation
levels associated with H. hepaticus-induced colitis in lL-10 deficient mice were
decreased by pre-treatment with L. reuteri 6798, a strain that had previously
been shown to reduce TNF levels in vitro (48). Interestingly, the colonization
levels of H. hepaticus were not affected, suggesting that immunomodulation may
be the main mechanism of action observed in this study. These animal studies
demonstrating a prevention or reduction of colitis with administration of L. reuteri
indicates that certain strains may be able to prevent or alleviate colonic

inflammation associated with inflammatory bowel disease (IBD).

A one-month clinical trial investigated the effect of administration of a
combination of L. reuteri RC-14 and L. rhamnosus GR-1 to individuals with IBD
(either Crohn’s or ulcerative colitis) or healthy control subjects. Different effects
of the probiotics were observed in both groups of individuals. One effect
observed was an increase in CD4+ CD25high T cells in the IBD patients; this

putatively immunosuppressive shift could help play a role in promoting and

29

maintaining remission in these patients (6). It is intriguing to note that probiotic

effects may be altered in response to the health of the host.

Hypercholesterolemia. Probiotics are also currently being investigated for the
ability to lower serum cholesterol levels in the host. L. reuteri CRL 1098 has been
shown to not only decrease cholesterol levels in mice, but also to prevent the
development of hypercholesterolemia (72, 73). In the first study,
hypercholesterolemic mice were fed L. reuteri CRL 1098 for seven days. After
this treatment, a significant reduction in total cholesterol, along with a reduction in
triglyceride levels and an increased ratio of HDL (high density lipoprotein) to LDL
cholesterol was observed. The authors suggest that these observed effects may
be due to either lower levels of intestinal absorption of cholesterol or higher
catabolism of lipids (73). One other consideration would be the possibility that
bile salt hydrolase activity of the bacteria resulted in the lower cholesterol levels
(7), as deconjugated bile acids are more likely to be excreted, requiring the host

to pull from serum cholesterol levels to synthesize new bile acids.

Colic. L. reuteri ATCC 55730 has even been investigated as a probiotic for the
alleviation of colic, an infantile disorder marked by excessive crying and irritability
of unknown cause. When compared in a clinical trial to simethicone (a common
colic treatment typically found to not be very effective), a reduction in crying time
was reported for the probiotic-treated group over the 28-day course of the study.

Crying time were reported at 51 minutes per day in the probiotic-treated group

30

and 145 minutes per day in the simethicone-treated group at the 28 day point,

suggesting that probiotics may alleviate colicky symptoms (61 ).

General health. Weizman et al (86) demonstrated that children in a daycare
setting that received L. reuteri not only had significantly fewer episodes of fever
and diarrhea than a control group, but also had significantly less clinical visits,
daycare absences, and antibiotic prescriptions. In another study where L. reuteri
ATCC 55730 was given to Swedish workers, improved health was found in the
probiotic-treated group. The probiotic-treated group had less sick leave due to
respiratory or gastrointestinal infections, and also had a lower frequency of sick
days (measured as the number of sick days relative to the number of work days)

than the control group (77).

Oral health. L. reuteri has also been included in studies involving oral health, in
part because of the potent antimicrobial activities of many of the strains. Many of
the studies have involved determining the effects of probiotic administration on
the levels of Streptococcus mutans, one of the main contributors to tooth decay.
L. reuteri ATCC 55730 has been shown to have in vitro inhibitory activity against
S. mutans; this antagonistic effect was also observed in human volunteers fed a
yogurt product containing the strain. In these volunteers, a significantly lower
population of S. mutans was isolated from the oral cavity, suggesting a
decreased risk of caries (46). Caglar has also demonstrated the effectiveness of

this strain by showing a significant reduction in salivary mutans streptococci

31

levels in volunteers that consumed L. reuteri ATCC 55370 in straw, tablet, or

gum form (9, 10).

Although the research in the probiotic field has increased exponentially over the
past twenty years or so, there is still much that needs to be investigated. The
research at this point includes many in vitro studies investigating survival of
stresses that the bacteria will be exposed to in the gastrointestinal tract, the
ability to inhibit pathogens, and the ability to modulate the host immune
response. In vivo studies are investigating a wide range of disorders including
bacterial infections, allergies, and gastrointestinal disorders. Although various
strains of L. reuten' have demonstrated effectiveness in various in vitro and in
vivo studies as discussed in this review, there are still large areas of research
that need to be conducted. For example, there appears to be wide ranging
differences between the physiology and effects of particular strains within the
species, and very little has been determined about the actual interactions

between L. reuteri and the host.

This thesis focuses on characterization of different aspects Of L. reuteri
physiology that may contribute to the probiotic potential of the species.
Micorarray and mutational analysis of the bile stress response of L. reuteri ATCC
55730 identified several genes that play significant roles in either survival or
growth of this strain in the presence of bile (Chapter 2). These genes may
contribute to survival of the strain during passage through the host

gastrointestinal tract. The study also identified a putative matrix metalloprotease

32

gene that is up-regulated in the presence of bile, but does not play a role in
survival or growth of the strain in bile. The function of this gene is of particular
interest, as it shows significant homology to eukaryotic matrix metalloproteases
which are known to play important roles in the homeostasis of the gastrointestinal

tract (Appendix C).

The bile salt hydrolase activity in L. reuteri ATCC PTA 6475 was also
investigated. The bacterial function Of this activity has yet to be elucidated,
although the research presented in this thesis suggests that it may be involved in
resistance to bile under acidic conditions (Chapter 3). An understanding of the
role this activity plays in survival of the bacteria during passage through the
gastrointestinal tract is important, as the activity has been proposed to also affect
the host. The proposed effects on the host range from a (beneficial lowering of
serum cholesterol levels to putatively harmful activation of carcinogens or

malabsorption of fats and nutrients.

Finally an investigation of the potential immunomodulatory role of lactobacillic
acid, a cyclopropyl fatty acid that is specific to certain strains of L. reuteri at
particular stages of growth is included. Certain strains of L. reuteri have been
demonstrated to suppress TNF production from LPS-activated macrophages in
vitro. This activity is proposed to be due to secreted molecules produced by
these strains at early stationary to late stationary phases of growth. The

production of lactobacillic acid is shown to be specific to strains with this

33

immunomodulatory activity, and the presence of this compound is shown to
correlate with the growth stages during which the immunomodulatory compound

is produced (Chapter 4).

34

10.

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Valeur, N., P. Engel, N. Carbajal, E. Connolly, and K. Ladefoged. 2004.
Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730

in the human gastrointestinal tract. Appl. Environ. Microbiol. 70:1176-
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Van Coillie, E., J. Goris, l. Cleenwerck, K. Grijspeerdt, N. Botteldoorn,
F. Van lmmerseel, J. De Buck, M. Vancanneyt, J. Swings, L. Herman,
and M. Heyndrickx. 2007. Identification of lactobacilli isolated from the
cloaca and vagina of laying hens and characterization for potential use as
probiotics to control Salmonella Enteritidis. J. Appl. Microbiol. 102:1095-

1 106.

Vesterlund, S., M. Karp, S. Salminen, and A. C. Ouwehand. 2006.

Staphylococcus aureus adheres to human intestinal mucus but can be
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81.

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84.

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Wall, T., K. Bath, R. A. Britton, H. Jonsson, J. Versalovic, and S.
Roos. 2007. The early response to acid shock in Lactobacillus reuteri

involves the CIpL chaperone and a putative cell wall-altering esterase.
Appl. Environ. Microbiol. 73:3924-3935.

Walter, J., P. Chagnaud, G. W. Tannock, D. M. Loach, F. Dal Bello, H.
F. Jenkinson, W. P. Hammes. and C. Hertel. 2005. A high-molecular-
mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB)
contribute to the ecological performance of Lactobacillus reuteri in the
murine gut. Appl. Environ. Microbiol. 71:979-986.

Walter, J., D. M. Loach, M. Alqumber, C. Rockel, C. Hermann, M.
Pfitzenmaier, and G. W. Tannock. 2007. D-alanyl ester depletion of
teichoic acids in Lactobacillus reuteri 100-23 results in impaired

colonization of the mouse gastrointestinal tract. Environ. Microbiol.
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Walter, J., C. Schwab, D. M. Loach, M. G. Ganzle, and G. W. Tannock.
2008. Glucosyltransferase A (thA) and inulosucrase (lnu) of Lactobacillus
reuteriTMW1.106 contribute to cell aggregation, in vitro biofilm formation,

and colonization of the mouse gastrointestinal tract. Microbiology 154:72-
80.

Wang, 8., H. Wei, J. Yuan, Q. Li, Y. Li, N. Li, and J. Li. 2008.
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Whitehead, K., J. Versalovic, S. Roos, and R. A. Britton. 2008.
Genomic and genetic characterization of the bile stress response of
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74:1812-1819.

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43

CHAPTER 2

GENOMIC AND GENETIC CHARACTERIZATION OF THE BILE STRESS

RESPONSE OF PROBIOTIC LACTOBACILLUS REUTERI ATCC 55730

These results have been published in the article: Whitehead, K., J. Versalovic, S.
Roos, and RA. Britton. 2008. Genomic and genetic characterization of the bile
stress response of probiotic Lactobacillus reuteri ATCC 55730. Appl. Environ.

Microbiol. Vol. 74, pgs 1812-1819.
INTRODUCTION

The idea that bacteria could benefit human health was postulated almost one
hundred years ago by Elie Metchnikoff (23). Recently the use of probiotics,
which are defined as “live microorganisms which when administered in adequate
amounts confer a health benefit on the host” (10), has become increasingly
popular. Probiotic microorganisms are currently being investigated for many
possible health benefits in many different ailments including inflammatory bowel
disease, diarrhea and hypercholesterolemia (9, 33, 40, 46). The mechanisms
through which probiotics confer their beneficial effects are mostly unknown;
examples of current theories include immunomodulation of the host by the

synthesis of immunomodulatory compounds, the production of antimicrobial

44

compounds that inhibit pathogen growth, and large-scale alterations of the

microbiota (28, 31).

Probiotic bacteria encounter a variety of stresses that need to be overcome to
remain viable. For example, many bacteria are packaged into food products
such as yogurt and fermented milk, which exposes them to temperature and
osmotic stress. After ingestion, probiotics must be able to survive the extreme
acidic conditions in the stomach and the detergent properties of bile acids in the
small intestine. Bile acids are amphipathic molecules that are synthesized from
cholesterol and play an important role in the digestion of fats and absorption of
fat-soluble vitamins. The concentration of bile acids ranges from 0.2 to 2% in the
human small intestine and fluctuates based on the amount of fat intake in the diet
(14). Bile acids have potent antimicrobial activity against many microbes and are
known to cause damage to cells that are considered to be bile resistant, most
likely via disruption of the membrane and cell wall. The resistance mechanisms
of gram-negative bacteria are fairly well-characterized; these mechanisms
include protection by the hydrophobic outer membrane and utilization of efflux
pumps to expel bile salts that do enter the cell (13). The resistance mechanisms
of gram-positive organisms, which in general are less bile-resistant than gram-

negative bacteria, are less well understood.

Lactic acid bacteria, particularly lactobacilli, are the genus most commonly used

as probiotics, in part because of their safe usage in food production. Potential

45

new probiotic strains should include several important characteristics; they
should be of human origin, non-pathogenic, able to remain viable in the
gastrointestinal tract for at least short periods of time, and be resistant to various
stresses (9). In the GI tract the main sources of antimicrobial stress are the low
pH encountered in the stomach and the detergent like properties of bile acids
found in the small intestine. Although the precise mechanisms by which bile
acids cause cell death are not understood, their chemical nature indicates they
will be able to solubilize membranes and cause significant membrane damage.
This is supported by genetic and genomic studies in a variety of different species
that show the main response of gram-positive organisms to bile exposure
appears to be alteration of the cellular envelope. Isolation of bile-sensitive
mutants of Enterococcus faecalis and Listeria monocytogenes identified genes
mainly involved in maintenance and synthesis of the cell membrane and wall, as
well as genes involved in general stress responses (2, 16). Microarray analysis
of Lactobacillus plantarum and Lactobacillus acidophilus identified expression
changes in genes whose product is found in the cell envelope (6, 25). In addition
there is also microscopic evidence supporting the role of bile in alteration of the
cellular envelope. Bron et al. demonstrated that cultures of L. plantarum cells
exposed to bile contained some shrunken cells and cells that tended to clump
together and had rough surfaces (5), while bile exposure also caused the
appearance of shrunken and empty cells in cultures of Propionibacterium

freudenreichii (1 7).

46

Lactobacillus reuteri is a species with a broad host range, with isolates
originating from many different species including humans, pigs, chickens, dogs,
mice, and hamsters (7). L. reuteri is also considered to be indigenous to the
human GI tract (27). L. reuteri ATCC 55730, a strain currently marketed for
probiotic usage, has been demonstrated in clinical trials to be effective against
diarrhea in children, as well as to alleviate colic in infants (26, 30, 45). In
addition, consumption of L. reuteri ATCC 55730 reduced the number of sick days
taken by workers in a large trial in Sweden (38). How these benefits are
achieved at the molecular level is still unknown. L. reuteri ATCC 55730 is known
to produce a broad-spectrum antimicrobial compound, reuterin, by metabolism of
glycerol under anaerobic conditions (36). Based on the observation that this
strain is able to survive in the human duodenum and ileum (41), it is an

appropriate organism to use in the investigation of bile resistance mechanisms.

This research investigated the gene expression response of L. reuteri ATCC
55730 to bile exposure and has begun to uncover the mechanisms this strain
uses to survive and grow in the presence of bile. Microarray experiments were
conducted to determine the gene expression profiles of cells upon initial bile
exposure (bile shock) and cells that had resumed growth in the presence of bile
(bile adaptation). Based on the microarray results, nine genes were chosen for
mutational analysis. These results indicate some of the mechanisms important in

the bile shock and adaptation responses of L. reuteri ATCC 55730 in vitro and

47

may provide a further understanding of characteristics important for survival in

the gastrointestinal tract.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterial strains and plasmids
used in this study are listed in Table 2.1. All liquid cultures of lactobacilli were
grown under microaerobic conditions (2% 02, 5% C02, balanced with N2) in MRS
broth (BD Difco) at 37°C, unless othen~ise specified. All plate cultures of
lactobacilli were grown under anaerobic conditions using the GasPack EZ
Anaerobe Container system (BD Difco) at 37°C, unless othenrvise specified.
Lactobacillus reuteri strains containing pVE6007 were grown at 35°C. All E. coli
was grown under aerobic conditions at 37°C in LB broth (BD Difco). When
specified, drugs were added to the following concentrations: 10 ug/ml (L. reuteri)
or 400 ug/ml (E. coli) erythromycin, 10 pig/ml chloramphenicol, and 40 pg/ml
kanamycin. L. reuteri mutants (containing the p0Rl28 disruption) were always
grown in the presence of 10 ug/ml erythromycin. Dehydrated bovine bile/ox gall
(Sigma) was resuspended in MRS broth to make a 50% weight/volume solution.
This mixture was sterilized by autoclaving and stored at 37°C for up to four

weeks.

48

Table 2.1. Bacterial strains and plasmids used for this study.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Strain or Reference
plasmid Description or source
Strains
E. coli strain containing a chromosomal copy
E01000 of the pWV01 repA gene; Kanr (15)
L. reuteri strain isolated from human breast Biogaia, AB,
ATCC 55730 milk Sweden
L. reuteri ATCC 55730 clpL (lr1864) mutant,
PRB190 Emr (42)
L. reuteri ATCC 55730 putative esterase
PRB188 (lr1516) mutant, Emr (42)
lr0085::pKW01 in an ATCC 55730
PRB167 background, Emr This study
L. reuteri ATCC 55730 clpE (lr0004) mutant,
PRB186 Emr Stefan Roos
lr1265::pKW02 in an ATCC 55730
PRB126 background, Emr This study
lr1584::pKW03 in an ATCC 55730
PRB130 background, Emr This study
lr1291::pKW04 in an ATCC 55730
PRB163 background, Emr This study
lr1351::pKW05 in an ATCC 55730
PRB125 background, Emr This study
lr1706::pKW06 in an ATCC 55730
PRB114 background, Emr This study
Plasmids
Cmr repA-positive temperature-sensitive
pVE6007 derivative of pWV01 (19)
p0Rl28 Emr repA-negative derivative of pWV01 (15)
pKW01 p0Rl28 + 203 bp insert from lr0085 This study
pKW02 ORI28 + 303 bp insert from Ir1265 This study
KW03 gORI28 + 331 bp insert from lr1584 This study
pKW04 p0Rl28 + 310 bp insert from lr1291 This study
pKW05 p0Rl28 + 152 bp insert from lr1351 This study
pKW06 p0Rl28 + 240 bp insert from lr1706 This study

 

49

 

RNA isolation. For each of five biological replicate experiments, a culture of L.
reuteri ATCC 55730 was grown in MRS broth to an optical density at 600 nm
(0.0.600) approximately equal to 0.5. Upon reaching this stage in growth, 0.5%
oxgall was added to the culture. At the correct time points, 5 ml samples were
collected from the culture and immediately mixed with an equal part of ice-cold
methanol. Cell pellets were collected by centrifugation, washed with STE buffer
(6.7% sucrose; 50 mM Tris-Cl, pH 8.0; 1 mM EDTA), and resuspended in STE
buffer containing 0.25 units/pl mutanolysin (Sigma). Cells pellets were then
incubated at 37°C for 20 minutes. RNA was then isolated using the Qiagen

RNeasy Kit according to manufacturer’s instructions.

Microarray experiments. Long oligonucleotides (60-mers) were designed and
synthesized for 1864 open reading frames from a draft genome sequence of L.
reuteri ATCC 55730 (1) and 15 open reading frames encoding known
extracellular proteins from L. reuteri DSM 20016 (43) using OligoArray 1.0
Software. Six control 60-mer oligonucleotides were also included. These controls
are identical to DNA sequences from E. coli genes (yacF, ybaS, yciC, yfiF, yng,
and ych) and have no sequence similarity to the L. reuteri genome. Once
synthesized the oligonucleotide concentrations were normalized to a
concentration of 25 uM and spotted onto Corning UltraGAPS-ll slides using an
OmniGrid Robot (GeneMachines). Each gene was represented once on the
microarray. All six of the control spots were represented 8 times on the array,

once in each subgrid. Oligonucleotide design, synthesis, and array construction

50

were performed at the Research Technology Support Facility at Michigan State
University, East Lansing, MI, USA. RNA isolation, labeling, and hybridization
were carried out essentially as previously described (39, 42). (Information
regarding the microarray platform can be found at NCBls Gene Expression
Omnibus (GEO, http://www.ncbi.nlm.nih.g)v/geo/) under GEO platform number

GPL6366).

Five biological replicates were performed for each of the two sets of microarray
experiments. In addition, technical replication was achieved by switching the
dyes used for labeling each biological replicate. Therefore, each RNA sample
was subjected to two hybridizations and values used for subsequent data
analysis were averages of the dye swap values. The first set of experiments,
referred to as the bile shock experiments, compared the gene expression profiles
of cells before exposure to 0.5% bile to those that had been exposed for 15
minutes. The second set of experiments, referred to as the bile adaptation
experiments, compared the gene expression profiles of cells before exposure to

0.5% bile to those that had begun growing again after exposure (Figure 2.1).

51

 

0.1 -

0.0.600

 

 

 

0.01 I I I T ‘T I I
0 100 200 300 400 500 600 700
time (min)

 

 

 

Figure 2.1. Representative growth curve of L. reuteri ATCC 55730 used for
microarray experiments (arrows represent time points where samples were taken
for RNA isolation). Open arrows represent samples for bile shock experiments;
filled arrows represent samples for bile adaptation experiments. 0.5% oxgall was
added at 250 minutes.

Microarray data was analyzed using iterative outlier analysis with three iterations
as previously described (4, 39). Briefly, iterative outlier analysis calculates the
geometric mean and standard deviation of the entire dataset. Differentially
expressed genes (outliers) were selected as being more than 2.5 standard
deviations away from the mean of the population. To identify additional

differentially expressed genes in the dataset. the outliers were removed and the

geometric mean and standard deviations were recalculated and any genes that

52

were more than 2.5 standard deviations from the mean were identified as

differentially expressed.

Mutant construction. Mutants were created using the system developed by
Russell and Klaenhammer (29) and modified for use in L. reuteri by Walter et al
(44). Briefly, 200 - 300 bp from the gene of interest were PCR-amplified from L.
reuteri ATCC 55730 and cloned into pORl28. The plasmid with the insertion was
then transformed into E. coli EC1000, a carrier strain that contains the RepA
protein needed for pORl28 to replicate; the transformed cells were grown in the
presence of erythromycin and kanamycin (EC1000). pORl28 with the insertion
was then extracted and transformed into L. reuteri ATCC 55730 cells containing
pVE6007. pVE6007 is a helper plasmid that provides RepA (also allowing
p0Rl28 to replicate). L. reuteri cells containing both plasmids were grown
aerobically without shaking at the permissive temperature of 35°C in the
presence of chloramphenicol and erythromycin for 18 hours. This culture was
then diluted 1:200 and grown aerobically in the presence of erythromycin without
shaking at 45°C for 8-24 hours. The 45°C culture was then plated onto MRS +
erythromycin plates and incubated at 45°C for 24 hours. Isolated colonies were
then obtained by streaking onto fresh IVIRS + erythromycin plates and incubated
at 45°C for another 24 hours to ensure loss of pVE6007. Individual colonies
were then selected for integration of pORl28 based on erythromycin resistance
and screened for chloramphenicol sensitivity to confirm loss of pVE6007 at 37°C.

Colonies that had lost pVE6007 were then screened for the correct insertion by

53

PCR-amplification of both flanking regions (one primer annealing to the
chromosome outside of the region cloned into pORl28 and one primer annealing
to pORl28) and confirmation of the absence of the correctly sized wild-type gene

(also through PCR). PCR primers are available by request.

Bile stress assays. To determine levels of bile resistance for the mutants, the
percent survival of wild-type and mutant cultures was determined after 30
minutes of exposure to 0.3% bile. In short, cultures were grown under
microaerobic conditions in MRS at 37°C to an O.D.500 = 0.5. Samples were
taken and colony counts were determined by dilution plating. 0.3% oxgall was
then added to each culture, and after 30 minutes, colony counts were again
determined. The before bile and after bile colony counts were used to determine
percent survival for each strain. All growth curves and viability plating were
performed three times for each strain, with the exception of the wild-type

experiments, which were repeated eight times.

RESULTS

Lactobacillus reuteri ATCC 55730 is able to grow in physiologically relevant
concentrations of bile. One important characteristic for probiotic bacterial
strains is the ability to remain viable during passage through the gastrointestinal
tract, including the ability to overcome exposure to bile stress in the small

intestine. Growth experiments were conducted to determine the response of L.

54

reuteri ATCC 55730 to physiological concentrations of bovine bile. In general,
when 0.05 to 0.1% bile was added to an early or mid-log culture (0.0600 = 0.2 or
0.5), the culture continued growing, although at a slightly reduced rate. ' The
doubling time of the culture would slow from 38 minutes before the addition of
bile to 50 minutes after the addition of bile. When concentrations of bile ranging
from 0.3% to 5% were added, we observed a period of growth arrest followed by
a resumption of growth. However the doubling time in the presence of these

higher bile concentrations was 3-4 times slower than prior to treatment with bile.

The growth-phase of L. reuteri cells also influenced the ability of bile to affect cell
growth and viability. Early log-phase cells (ODsoo = 0.2) were the most resistant
to the effects of bile treatment. Addition of bile at later stages of log-phase
growth and early stationary phase indicated that cells (become more susceptible
to bile as the culture density increases (as measured by a decrease in the optical
density and cell viability of the culture after the addition of bile). Active growth
appears to be required for this effect as late stationary phase cultures were

completely resistant to bile stress, even at concentrations of 5%.

Microarray analysis of genes involved in bile shock and adaptation. We
used DNA microarrays to characterize both the bile shock response and bile
adaptation response of L. reuteri. When cells encounter stress they often
respond by altering their gene expression program to effectively counteract

stress-induced damage. Because L. reuteri exhibits a biphasic response to bile

55

exposure we measured the global RNA profiles of cells that were paused for
growth (which we denote as bile shock) and cells that had resumed growth in the

presence of bile (bile adaptation).

i. Bile shock. In order to determine the genes involved in the bile shock
response, microarray experiments were carried out to compare the gene
expression profiles of mid-log cells that had not been exposed to bile to cells that
had been exposed to 0.5% bile for 15 minutes. Eighty-eight genes were found to
have significant expression changes, with 45 genes over-expressed and 43
genes under-expressed after 15 minutes of bile exposure. The majority of under-
expressed genes are classified as being involved in substrate transport and
metabolism, which is expected due to the lack of growth observed upon
exposure to bile (Table 2.2). The over-expressed genes are found in a wide
variety of classes and several have known roles in adaptation to other types of

stresses (see below and discussion).

56

Table 2.2. Classesa of genes differentially expressed
during the first 15 minutes of exposure to 0.5% bile.

 

 

Number of genes Number of genes
over-expressed under-expressed
Gene classification during bile shock duringbile shock
Energy production and
conversion 0 4
Cell division and
. . b
envelope biogeneSIs 1 3
Substrate transport and
metabolismC 4 21
Translation, ribosomal
structure and biogenesis 2 2
Transcription 8 1
Replication,
recombination and repair 3 3
Posttranslational
modification, protein
turnover, chaperones 6 2
Defense mechanisms 3 0
Unknown functions 18 7
Total 45 43

 

aGenes were classified based on COG domains found in the protein sequence
through a search of the JGI Integrated Microbial Genomes database.

b . . . . .
This class represents two COG categories: cell cycle control, cell lelSlOl‘I, and
chromosome partitioning and cell wall/membrane/envelope biogenesis.

CThis class represents multiple COG categories that include transport and
metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids,
inorganic ions, and secondary metabolites.

ii. Bile adaptation. The expression profiles of mid-log cells that had not been
exposed to bile were also compared to the profiles of cells that had resumed

growth in the presence of 0.5% bile. After analysis of this set of array

experiments, 84 genes were found to have significant expression changes, with

57

17 being over-expressed during the adaptation stage and 67 being under-
expressed. Again, the majority of under-expressed genes during growth in bile
are classified as being involved in substrate transport and metabolism; in
addition, genes involved in energy production, translation and ribosome structure
and biogenesis, and genes of unknown function are also under-expressed. Most
of these changes are likely due to the dramatic reduction in growth rate of L.
reuteri cells grown in the presence of bile. There were 17 genes that were
significantly over-expressed during growth in bile; over half of these genes are
annotated as having unknown functions (Table 2.3). The expression patterns of
a subset of genes that were differentially expressed were found to overlap
between the two sets of microarray experiments, with eight genes being over-
expressed and 12 genes being under-expressed during both bile shock and
adaptation. (A full description of the differentially expressed genes discovered in
all of the microarray experiments can be found in Supplementary Tables 2.7 to
2.10 (located at the end of this chapter). A complete dataset of microarray data
can be found at NCBls Gene Expression Omnibus (GEO,
http://www.ncbi.nlm.nih.gov/qeol) under GEO Series accession number

GSE10155).

58

Table 2.3. Classesa of genes differentially
expressed during growth in the presence of 0.5%

 

 

bile.
Number of genes Number of genes
over-expressed under-expressed
Gene classification during bile adaptation during bile adaptation
Energy production and
conversion 0 11
Cell division and envelope
biogenesis 0 4
Substrate transport and
metabolism6 5 23
Translation, ribosomal
structure and biogenesis 0 11
Transcription 2 1
Replication,
recombination and repair 0 4
Intracellular trafficking,
secretion, and vesicular
transport 0 2
Signal transduction
mechanisms 0 2
Defense mechanisms 1 0
Unknown functions 9 9
Total 17 67

 

aGenes were classified based on COG domains found in the protein sequence
through a search of the JGI Integrated Microbial Genomes database.

bThis class represents two COG categories: cell cycle control, cell division, and
chromosome partitioning and cell wall/membrane/envelope biogenesis.

cThis class represents multiple COG categories that include transport and
metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids,
inorganic ions, and secondary metabolites.

Mutations in three genes, Ir0085, Ir1516, and Ir1864, decrease the ability of

cells to survive bile shock. Bile salts have been proposed to cause a wide-

range of cellular effects, including cell wall or membrane damage, DNA damage,

59

protein denaturation, oxidative stress, and low intracellular pH (3). Several
genes were chosen for mutation based on their proposed functions in adapting to
a variety of stresses. Disruptions were created using the pVE6007/pORl28
system in nine genes that were found to be significantly over-expressed during
the bile exposure of L. reuteri ATCC 55730 (Table 2.4). Two Clp chaperones
(lr0004 [clpE]) and lr1864 [clpL]) were disrupted; Clp chaperones have been
implicated in the heat shock response of Bacillus subtilis, as well as other gram-
positive organisms (8, 11). The dps gene (lr1706) and a putative esterase
(lr1516) were also disrupted to investigate the proposed oxidative stress and cell
wall damage effects of bile. The putative esterase (lr1516) belongs to a cluster
of orthologous genes (COG) that includes B-Iactamase class C and other various
penicillin-binding proteins (20). Three other genes of unknown function were
also chosen for disruption: lr1291, a putative metalloproteinase, lr1351, a
conserved membrane protein, and Ir0085, a gene of unknown function that
appears to be specific to the species L. reuteri. Finally, two multidrug resistance
transporters were disrupted (Ir1265 and lr1584). E. coli has the ability to actively
pump bile salts out of the cell, and efflux pumps, similar to drug resistance

transporters, have been found to have a major role in this activity (37).

60

Table 2.4. Fold gene expression changes in the
presence of bile for genes chosen for disruption.

 

 

Fold-
changeb Fold- b
, during change
Aegessmn bile during bile
Gene no. Annotation shock adaptation
lr0004 EF421856 Clp chaperone (ClpE) 5 1.4
lr0085 00233699 Hypothetical protein 3.3 2.5
Multidrug resistance protein (ABC
Ir1265 EU038268 transporter family) 3.9 1.5
lr1291 AY970991 Metalloproteinase 2.8 1.6
Conserved membrane protein of
[H351 00233687 unknown function 3 4.1
Ir1516 D0219970 Putative esterase 7.3 2.8
Multidrug resistance protein (Major
lr1584 EU038252 facilitator superfamily) 1.6 2.2
lr1706 EU038255 Dps 2.6 2.1
Ir1864 D0219976 Clp chaperone (CIpL) 3.2 1.4

 

a . .
GenBank accessron numbers are prOVIded.

bFold-changes in bold were found to be significantly
different based on the outlier analysis.

Each of the nine mutants was subjected to treatment with various concentrations

of bile to determine which mutants are defective in surviving bile shock. Based

on these preliminary experiments, the survival of six of these mutant strains were

quantitated and compared to the viability of wild-type cells after 30 minutes of

0.3% bile exposure; the other three mutants (lr0004, lr1291, and lr1351) showed

no defect in bile shock and were not further tested. Using a Student’s t-test, it

was determined that the survival rate after 30 minutes of bile exposure for three

of the mutants (lr1864, lr1516, and lr0085) was significantly different from that of

61

wild-type (p<0.001).

Mutations in dps and the two multidrug resistance

transporters (lr1706, lr1584, and Ir1265) did not have a significantly different

survival rate from wild-type cells (Table 2.5; Figure 2.2).

Table 2.5. Effects of 0.3% bile exposure on cell viability and final
culture density.

 

 

Disrupted Accession Percentb
gene no.a Annotation survival 0.06000
Ir0085 D0233699 Unknown 16 i 3%* 17:09
Multidrug resistance
protein (ABC transporter *
Ir1265 EU038268 family) 66 1 23% 1.0301
lr1516 DQZ19970 Putative esterase 8 1 2%* 2110.1
Multidrug resistance
protein (Major facilitator *
lr1584 EU038252 superfamily) 69 i 32% 0.6100
lr1706 EU038255 Dps 55 i 21% 13:03
lr1864 D0219976 Clp chaperone (CIpL) 3 1 1%* 1.6302
Wild-type cells 54 i 16% 16:02

 

a . .
GenBank acceSSIon numbers are prOVIded.

b . .
(Percent survwal 30 minutes after exposure.
Data is presented as percent survival 1 standard

deviation.

cData is presented as 0.0500 1 standard deviation
after growth had ceased.

*p<0.001 compared with wild-type.

62

120
100 1-

80

60 I
40 I " _,

20

*
1:
°k
m
0 ......4 __.r_. .--.,----. we.-. ._-_..T-,.... 2.... .fi.

ATCC PRB190 PR8188 PRB167 PRBI14 PRB126 PRB130
55730

 

 

 

 

 

 

Percent survival

 

 

 

 

 

 

 

 

 

 

 

Strain

Figure 2.2. Comparison of survival after 30 minutes of exposure to 0.3% oxgall
for L. reuteri ATCC 55730 wild-type and PRB190 (Ir1864 - CIpL), PRB188 (lr1516
- putative esterase), PRB167 (lr0085 - unknown), PRB114 (lr1706 - Dps),
PRB126 (Ir1265 - multidrug resistance protein in the ABC transporter family), and
PRB130 (lr1584 - multidrug resistance protein in the major facilitator superfamily)
mutant strains. Cultures were plated onto MRS plates after bile exposure to
determine the number of viable cells. Error bars represent standard deviation.
*p < 0.001 compared with wild-type.

Mutations in a putative operon encoding a multidrug resistance protein and
a hypothetical protein decrease the ability of L. reuteri to adapt in the
presence of bile. When testing the mutant strains for survival in the presence of
bile, it was observed that the Ir1584 mutant did not adapt to grow in the presence

of bile, even after extended incubations (24 hours). The final culture density

63

obtained for the lr1584 mutant was found to be three-fold lower than that of the
wild-type strain. Additional experiments revealed that most of the other mutants
were not affected in their ability to adapt and grow in the presence of bile. The
exceptions were the Ir1265 mutant, which also obtains a lower culture density
than wild-type, and lr1516 mutant, which obtains a slightly higher culture density
(Table 2.5). The decreased ability of the Ir1584 and Ir1265 mutant strains to
adapt to the presence of bile suggests that multidrug resistance efflux pumps
play a role in this strain’s bile response. Efflux pumps have already been shown

to play important roles in the bile response of other bacteria (18, 34, 37).

The lr1584 gene is found in a putative operon with a gene encoding a conserved
hypothetical protein, lr1582. Ir1582 is also found to be significantly over-
expressed in the presence of bile; therefore, we were concerned about the
possible polar effects the disruption in lr1584 would have on the downstream
gene, lr1582. To distinguish between the effects of the Ir1584 mutation and the
possible polar effects on Ir1582, a separate mutant strain containing a disruption
in Ir1582 was created and tested for its ability to adapt in the presence of bile.
This strain also showed an adaptation defect; the final culture density of the
Ir1582 mutant was two-fold lower than that of wild-type cells. This demonstrates
that the adaptation defect seen in the Ir1584 mutant cannot be fully explained by
polar effects on the downstream gene, Ir1582, and suggests that both genes in

this operon play a role in L. reuteris adaptation to bile.

64

DISCUSSION

The ability of a bacterium to resist bile stress is one of the criteria often used in
the selection of a potential probiotic. Bile is a complex mixture of bile acids,
phospholipids, proteins, ions, and pigments that has potent antimicrobial
properties, particularly against gram-positive bacteria. In this study we have
identified several genes that participate in the ability of Lactobacillus reuteri
ATCC 55730 to tolerate bile shock and to resume growth in the presence of bile

(bile adaptation).

Stress responses activated in L. reuteri based on gene expression data.
The gene expression data indicate that membrane/cell wall stress, oxidative
stress, DNA damage, and protein denaturation occur when L. reuteri is exposed
to bile. Several of these pathways have been previously shown to be involved in
dealing with various forms of stress in other bacteria. First, the Clp chaperones
ClpE and CIpL are induced 15 minutes after bile addition as is their known
transcriptional regulator in other gram-positive organisms, CtsR. CtsR is a
repressor of multiple clp chaperones in Listeria monocytogenes and Bacillus
subtilis and also represses its own expression. Previous work has shown that
the induction of the CtsR stress regulon is transient with an initial peak of
expression under heat or salt stress that then is reduced after a period of time
(32). Consistent with this mode of regulation in other bacteria is the fact that we

observe that ctsR and the clp chaperones are overexpressed only during bile

65

shock and not during bile adaptation. c/pL was specifically required for L. reuteri
to resist bile shock while the clpE mutant did not survive significantly different
than wild-type cells. Repeated attempts to construct a mutation in the ctsR gene
were unsuccessful. Second, we also observed increased expression of dps, a
protein involved in several types of stress adaptation in Escherichia coli including
oxidative stress, irradiation, metal toxicity, heat stress, and pH stress (22, 24).
However, disruption of dps in L. reuteri did not significantly affect their ability to
survive bile shock or adapt to the presence of bile. Finally, two additional stress
response genes and one additional pathway were also induced. A homolog of
the gene glsZ4 (lr2108) was induced; G|324 was previously identified as a bile-
induced protein in Enterococcus faecalis. Subsequent genetic analysis indicated
it was required for the ability of E. faecalis to survive bile exposure, however no
molecular function for Gls24 is known (12). The gene lr1346 encodes a homolog
of the phage shock transcriptional regulator PspC, which is proposed to be
involved in sensing membrane stress during phage infection. Given that bile
likely induces membrane stress it is possible that lr1346 plays a role in bile stress
survival. Unfortunately we were unable to disrupt lr1346 due to limitations of our
gene knockout technology. Lastly, genes of the arginine deiminase pathway
were specifically induced during bile adaptation. This pathway has been

implicated in the ability to resist mild pH shock in bacteria (21).

The identification of lr1516, a putative esterase of the serine B-lactamase-Iike

superfamily, as a key enzyme in responding to bile stress suggests these cells

66

are experiencing cell envelope damage upon exposure to bile. Lr1516 contains
the signature SxxK active site motif associated with these enzymes, which also
include the D-alanyl-D-alanine carboxypeptidases. These enzymes are involved
in the breakdown and reorganization of peptidoglycan, and thus we expect that

lr1516 may play a similar role when adapting to bile and acid stress (42).

Bile adaptation. Because L. reuteri has been shown to colonize, at least
temporarily, the small intestine, we were interested in determining if L. reuteri can
thrive in the presence of bile. Our results demonstrate that L. reuteri can sustain
growth in the presence of bile concentrations as high as 5%. Interestingly the
data suggest a multidrug resistance transporter (lr1584) is required for this ability
to grow in the presence of bile, suggesting that removal of bile or another toxic
metabolite from the cytoplasm is required for growth. Lr1584 is a member for the
EmrB/QacA subfamily of the major facilitator superfamily of multidrug resistance
transporters. EmrB has previously been shown to play a role in bile resistance

and efflux of bile in Escherichia coli. (37).

Interestingly, lr1584 is found in an operon upstream of a conserved hypothetical
protein, Ir1582; this operon is conserved in many lactic acid bacteria and is over-
expressed during exposure of L. reuteri, L. acidophilus, and E. faecalis to bile
(25, 35). Due to the limited genetic tools available for use in L. reuteri, we were
not completely able to distinguish between the effects of disrupting Ir1584 and

possible polar effects this disruption may have on the downstream Ir1582. A

67

separate mutant strain with a disruption in Ir1582 was created and tested for bile
adaptation. The Ir1582 mutant does result in an adaptation defect, although it is
not as severe as the defect found in the lr1584 mutant strain. The final culture
density of the lr1584 mutant is approximately 3-fold lower than that of the wild-
type cells, while the final culture density of the Ir1582 mutant is approximately 2-
fold lower than wild-type cells. This suggests that both genes may contribute to
the adaptation defect that we have observed. Further investigation is required to

elucidate the specific role of each gene in bile adaptation.

Genes that provide protection in bile stress also protect against acid
stress. We identified three genes in L. reuteri that were induced by bile stress
and that significantly reduced their ability to survive bile shock when disrupted.
Two of these proteins have recently been shown to be induced by a strong
reduction in pH and are necessary for increased survival at low pH (42). Both
lr1864 (clpL) and lr1516 (putative esterase) play a role in surviving the initial

shock of acid and bile stress.

Indeed nearly one-third of the genes Wall et al. found to be differentially
regulated in acid stress conditions were also altered under bile stress conditions
(Table 2.6) (42). This indicates that once cells experience acid stress in the
stomach many of the important pathways for dealing with bile stress in the small

intestine will already be activated.

68

Table 2.6. Genes over-expressed or under-expressed during both 15 minutes
of bile exposure (0.5% oxgall) and 15 minutes of acid stress (pH 2.7) (42).

 

 

Accession
Gene no.8 Annotation
Over-expressed
lr0597 00219952 Thioredoxin domain-containing protein
Ir0922 00074860 Extracellular hydrolase
Ir1139 AY970988 Conserved intracellular protein of unknown function
lr1191 00219999 Conserved membrane protein of unknown function
Ir1468 00219968 Putative transcriptional regulator
lr1515 00074905 Unknown extracellular protein
lr1516 00219970 Putative esterase
lr1797 00219975 Phosphatidylglycerolphosphatase A and related proteins
Ir1864 00219976 CIpL ATPase with chaperone activity
Ir1937 00219979 Conserved intracellular protein of unknown function
lr1993 00219980 Putative transcriptional regulator
lr2045 00219981 Phage-associated protein
Under-expressed
lr0190 00219995 Transcriptional regulator
lr0195 00219996 Putative 5-formyltetrahydrofolate cyclo-ligase
Ir0382 AY971000 Putative branched-chain amino acid transport protein
Ir0733 00219954 Conserved intracellular protein of unknown function
lr0862 00219957 Asp-tRNAAsn/Glu-tRNAGIn amidotransferase C subunit
Ir1240 00219962 Recombinational DNA repair ATPase
Ir1297 00219963 Thymidine kinase
lr1432 00220005 Ribosomal protein S1
lr1434 00074902 Unknown extracellular protein

Conserved intracellular protein, MarZ, of unknown

Ir1628 00219972 function

 

a . .
GeneBank acceSSIon numbers are prOVIded.

Comparison of multiple genomic studies of bile stress in lactic acid

bacteria. Multiple genomic studies have now been completed that identified

genes important for bile tolerance in different species of Lactobacillus and

Enterococcus (5, 6, 25, 35). Although different culture conditions, types of bile,

and species were used for these studies, which resulted in a limited overlap in

the genes identified in these studies, there are some common themes that have

69

emerged. In both Lactobacillus plantarum and Lactobacillus acidophilus several
genes involved in the reorganization of the cell envelope were induced. Thus
dealing with membrane stress is a common theme that has emerged from these
three studies. In addition, the operon containing Ir1584 (multidrug resistance
transporter) and Ir1582 (unknown function) is also conserved as an operon in L.
acidophilus and Enterococcus faecalis. Interestingly, both homologs in E.
faecalis and L. acidophilus are also overexpressed when cells were exposed to
bile, indicating the function of this operon in bile adaptation may be conserved in
other lactic acid bacteria (25, 35). Lastly, clp proteases were identified in L.
acidophilus as being upregulated by bile stress as we found with clpL in our
study, further supporting that protein denaturation is one stress being

encountered by bile treated cells.

One common finding that is not easily explained is the reduction in gene
expression of recF, which encodes a protein that participates in the repair of DNA
damage during active DNA replication. Since bile has been implicated in
generating DNA damage, on the surface it seems that a reduction in the
expression of RecF would not be productive. However, recent evidence
indicates that RecF is predominantly utilized in DNA repair at replication forks
during active growth. The reduction in recF expression may simply indicate a

reduction in growth rate in the presence of bile.

7O

Although there were significant similarities in the transcriptional profiles between
the E. faecalis, L. acidophilus, L. plantarum, and L. reuteri responses to bile
treatment; overall there was much more discordance in the data than similarities.
The lack of concordance may be due to the different physiological strategies
utilized by these organisms to adapt to bile stress. In addition, the differences in
experimental strategy (the type of bile used, the way bile was administered, and
for how long) likely also played a significant role in the differences that were
noted. Bron et al. exposed L. plantarum to 0.1% porcine bile on plates and
looked at the response to three days of exposure, while Pfeiler et al. conducted
their L. acidophilus experiments in liquid media containing 0.5% oxgall with 30
minutes of exposure (6, 25). The use of purified bile acids or different sources of

bile will also have different effects on cell physiology (3).

Bile has been implicated as a potential signaling molecule that would indicate to
a bacterium that it had entered the small intestine. Such a signal could serve to
stimulate the organism to adapt its physiology to optimize growth and survival in
the GI tract. Several candidates from these experiments have now been
identified in vitro. Future work in relevant animal models will determine if the

strategies uncovered here are important for survival in vivo.

71

SUPPLEMENTAL INFORMATION

Table 2.7. Genes over-expressed after 15 minutes of exposure to 0.5%
oxgafl.

 

 

 

 

 

 

 

 

 

 

 

Accession Fold-
Gene name no.a Functional Classificationb change
Cell division and envelope biogenesisc
ABC-type transport system involved
in lipoprotein release permease
lr1816 00074937 component 3.4
Defense mechanisms
‘ Multidrug resistance ABC
transporter ATP-binding and
Ir1265 EU038268 permease protein 3.9
lr1516 00219970 Putative esterase 7.3
ABC transporter, ATP-binding
lr1817 EU038262 protein 2.3
Unknown function
lr0085 00233699 Hypothetical protein 3.3
Ir0540 EU038232 Conserved hypothetical protein 2.9
lr0542 EU038234 Conserved hypothetical protein 3.1
|r0543 EU038235 Cva family protein 2.3
lr0922 00074860 Extracellular hydrolase 2.2
Conserved membrane protein of
lr1 191 00219999 unknown function 3.2
Conserved intracellular protein of
lr1 139 AY970988 unknown function 2.5
Intracellular protein of unknown
lr1350 00233686 function 5.3
Conserved membrane protein of
lr1351 00233687 unknown function 3.1
lr1515 00074905 Unknown extracellular protein 13.6
lr1684 00857799 Conserved hypothetical protein 2.5
lr1740 EU038256 Acetyltransferase (EC 2.3.1 .-) 3.5
Conserved hypothetical protein,
lr1755 EU038257 putative pseudogene 2.5

72

 

Table 2.7 (cont’d)

Conserved intracellular protein of

 

 

 

 

 

 

 

Ir1937 00219979 unknown function 2.3
lr1990 00074949 Unknown extracellular protein 2.5
lr1994 EF421933 Conserved hypothetical protein 3.1
lr2045 00219981 Phage-associated protein 2.3
lr2108 00233697 Putative stress response protein 2.6
Posttranslational modification, protein turnover, and
chaperones
ATP-dependent clp protease ATP-
lr0004 EF421856 binding subunit clpE 4.9
Thioredoxin domain-containing
lr0597 00219952 protein 2.2
Ir1268 00857779 Hypothetical protein 2.4
lr1291 AY970991 Putative metalloproteinase gene 2.8
lr1788 EU038260 Thioredoxin 2.2
CIpL ATPase with chaperone
lr1864 00219976 activity 3.2
Replication, recombination, and repair
Holliday junction resolvase-like
lr0541 EU038233 protein 3.2
lr0685 EU421874 Transposase 3.3
DNA mismatch repair protein
Ir2132 EU038269 MutS2 2.5
Transcription
lr0783 EF421875 Transcriptional regulator ctsR 2.3
Transcriptional regulator, MarR
lr1264 EU038242 family 3.8
Stress-responsive transcriptional
Ir1346 EU038243 regulator PspC 2.7
Transcriptional regulator, TetR
lr1347 EU038244 family 4.3
Ir1468 00219968 Putative transcriptional regulator 3.1
lr1573 E7038251 Transcription regulator, Aan-type 2.3
Ir1815 EU038261 Transcriptional regulator 2.5
lr1993 00219980 Putative transcriptional regulator 2.2

 

73

 

Table 2.7 (cont’d)

 

Translation, ribosomal structure and biogenesis

 

Ribosome-associated protein Y
lr1067 EF421891 (PSrp-1) 2.4

Ribosomal large subunit
pseudouridine synthase, RIuD
lr1786 E7038259 subfamily 3.1

 

Substrate transport and metabolismd

 

 

Transcriptional regulator, GntR

lr0621 EU038237 family / aminotransferase 2.2
Thiamine pyrophosphate-requiring

lr1 1 17 00857776 enzymes 2.3
DPS (DNA protection during

Ir1706 EU038255 starvation) protein 2.6
Phosphatidylglycerolphosphatase A

lr1797 00219975 and related proteins 2.5

 

a GenBank accesion numbers are provided.

b Genes were classified based on COG domains found in the protein
sequence through a search of the JGI Integrated Microbial Genomes
database. ‘

c This class represents two COG categories: cell cycle control, cell division,
and chromosome partitioning and cell wall/membrane/envelope biogenesis.

d This class represents multiple COG categories that include transport and
metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids,
inorganic ions, and secondary metabolites.

74

 

Table 2.8. Genes over-expressed during growth in the presence of 0.5%

 

 

 

 

 

 

 

 

 

 

 

 

 

oxgafl.
Accession Fold-
Gene name no.a Functional Classificationb champ
Defense mechanisms
lr151 6 00219970 Putative esterase 2.8
Unknown function
Ir0085 00233699 Hypothetical protein 2.5
lr0890 00233682 Putative acetyltransferase 2.4
Ir1035 00857774 Conserved hypothetical protein 2.2
lr1348 00074898 predicted membrane protein 2.1
Intracellular protein of unknown
lr1350 00233686 function 5.4
Conserved membrane protein of
lr1351 00233687 unknown function 4.1
lr1515 00074905 Unknown extracellular protein 3.7
Ir1582 EF421918 Conserved hypothetical protein 2.8
Ir2108 00233697 Putative stress response protein 2.3
Transcription
Transcriptional regulator, TetR
lr1347 EU038244 family 2.3
lr1518 00233704 ArgR Arginine repressor 2.2
Substrate transport and metabolismc
lr1019 00857773 ArcC Carbamate kinase 2.0
ArgF Ornithine
lr1020 00233707 carbamoyltransferase 2.7
Ir1517 00233695 ArcA Arginine deiminase 2.6
Permease of the major facilitator
lr1584 EU038252 superfamily 2.2
DPS (DNA protection during
Ir1706 EU038255 starvation) protein 2.1

 

a GenBank accesion numbers are provided.

b Genes were classified based on COG domains found in the protein
sequence through a search of the JGI Integrated Microbial Genomes

database.

c This class represents multiple COG categories that include transport and
metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids,
inorganic ions, and secondary metabolites

75

 

Table 2.9. Genes under-expressed after 15 minutes of exposure to 0.5%
oxgafl.

 

ACCESSION FOId-

Gene name no.a Functional Classificationb change

 

 

 

 

. . . . C
Cell leISIOI‘I and envelope biogenesis

 

lr0548 EU038236 Cell division initiation protein 3.2
Capsular polysaccharide

lr0957 00857880 biosynthesis protein 1.9
S-adenosyl-methyltransferase

lr1629 EU038253 MraW 2 .4

 

Energy production and conversion

 

CitC Citrate lyase synthetase

lr0599 00233700 partial CDS 1.9
lr0600 00240820 Sch malic enzyme 2.1
lr1073 EF534266 Fumarase 2.0

PduA propanediol utilization
protein: putative

 

 

 

 

lr1882 00233726 microcompartment protein 2.9

Unknown function

lr0196 EU038226 Rhomboid family protein 1.9
Conserved intracellular protein of

lr0733 00219954 unknown function 2.7

lr1241 EF421898 Conserved hypothetical protein 3.0

lr1267 00074894 Unknown extracellular protein 2.1

lr1434 00074902 Unknown extracellular protein 2.0
Conserved intracellular protein,

Ir1628 00219972 MarZ, of unknown function 2.2

lr2134 EU038270 ComG operon protein 1 2.4

Posttranslational modification, protein turnover, and

chaperones

lr1373 EU038246 FeS assembly ATPase Squ 1.9

lr1774 EU038258 Glutaredoxin-like protein NrdH 3.0

 

Replication, recombination, and repair

 

 

ATPase related to the helicase
subunit of the Holliday junction

lr0044 EF421 958 resolvase 2.3
Nuclease subunit of the

lr0311 00857869 excinuclease complex 2.0
Recombinational DNA repair

lr1240 00219962 ATPase 3.4

 

76

 

Table 2.9 (cont’d)

 

Transcription

 

lr0190 00219995 predicted transcriptional regulator

 

Translation, ribosomal structure and biogenesis

 

Asp-tRNAAsn/Glu-tRNAGIn

 

 

 

lr0862 00219957 amidotransferase C subunit 2.6

lr1432 00220005 Ribosomal protein S1 2.4

Substrate transport and metabolismd
Spermidine/putrescine ABC

lr0137 EF537897 transporter, permase protein 3.4

lr0160 EF547651 Sugar kinase, ribokinase family 4.3

lr0187 EU038223 Dihydroneopterin aldolase 2.3
2-amino-4-hydroxy-6-
hydroxymethyldihydropteridine

lr0188 EU038224 pyrophosphokinase 2.0

lr0189 EU038225 GTP cyclohydrolase l 1.9
Putative 5-formyltetrahydrofolate

lr0195 00219996 cycIo-ligase 2.2
Cystathionine beta-lyase (EC
4.4.1.8) / Cystathionine gamma-

lr0324 EF421866 lyase (EC 4.4.1.1) 2.9
Putative branched-chain amino

lr0382 AY971000 acid transport protein 2.1
Permease of the major facilitator

Ir0848 EU038267 superfamily 2.1

Ir1297 00219963 Thymidine kinase 2.1
PduC propanediol dehydratase

lr1880 00233724 large subunit 2.0

, PduB propanediol utilization

Ir1881 00233725 protein 3.1

lr1959 00857828 HemA, Glutamyl-tRNA reductase 2.2

lr1960 00857829 Putative siroheme synthase 2.7

lr1961 00857830 CbiP, cobyric acid synthase 2.3

|r1963 00857832 Cbi0, cobalt transport protein 2.2
CbiB, cobalamin biosynthesis

lr1977 00857846 protein 2.0
CbiA, cobyrinic acid a,c-diamide

lr1978 00857847 synthase 2.2
CobD, L-threonine-0-3-phosphate

Ir1979 00857848 decarboxylase 3.2

77

 

 

Table 2.9 (cont’d)

Diacylglycerol kinase family

lr2131 EU038263 protein 2.4
ABC-type ribose transport system,
lr2133 EU038271 auxiliary component 2.3

 

a GenBank accession numbers are provided.

b Genes were classified based on COG domains found in the protein
sequence through a search of the JGI Integrated Microbial Genomes
database.

c This class represents two COG categories: cell cycle control, cell division,
and chromosome partitioning and cell wall/membrane/envelope biogenesis.

d This class represents multiple COG categories that include transport and
metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids,
inorganic ions, and secondary metabolites.

78

 

Table 2.10. Genes under-expressed during growth in the presence of

 

 

 

 

 

 

 

 

 

 

 

0.5% oxgall.
Accession Fold-
Gene name no.a Functional Classificationb change
Cell division and envelope biggenesisc
GidB predicted S-
adenosylmethionine-dependent
Ir0145 00219992 methyltransferase partial CDS 1.7
WcaA Glycosyltransferases
lr0740 00233678 involved in cell wall biogenesis 1.6
Capsular polysaccharide
lr0957 00857880 biosynthesis protein 1.7
UDP-MurNAc—pentapeptide
lr1645 EU038254 synthetase 1 .8
Energy production and conversion
CitC Citrate lyase synthetase
lr0599 00233700 partial CDS 2.1
lr0600 00240820 Sch malic enzyme 1.7
Fumarate reductase fiavoprotein
lr1074 EF534267 subunit (EC 1.3.99.1) 2.5
L-lactate dehydrogenase (EC
lr1075 EF421892 1.1.1.27) 2.8
Cytochrome bd-type quinol
lr1236 EF534271 oxidase, subunit 1 1.9
Ir1402 EF421914 ATP synthase subunit A 1.7
Ir1403 EF421915 ATP synthase subunit C 1.6
lr1404 EU038247 ATP synthase F0, B subunit 1.8
lr1405 EU038248 ATP synthase F1, delta subunit 1.7
lr1406 EU038249 ATP synthase F1, alpha subunit 1.7
COG3051 CitF Citrate lyase
lr1418 00233691 alpha subunit 1.9
Intracellular trafficking, secretion, and vesicular transport
COG0706 preprotein translocase
lr0252 00074813 subunit YidC 1 .6
COG0201 preprotein translocase
Ir0469 00074824 subunit SecY 1.8

 

79

 

Table 2.10 (cont’d)

 

Unknown function

 

Beta-phosphoglucomutase (EC
5.4.2.6) / GIucose-1-phosphate

 

 

 

 

 

 

 

 

 

80

lr1054 00466579 phosphodismutase (EC 2.7.1.41) 1.6
lr1487 00074904 Unknown extracellular protein 1.7
Ir2054 00219984 Phage head maturation protease 2.0
lr2057 00219986 Putative phage protein 1.7
lr2058 00219987 Putative phage protein 1.6
Ir2134 EU038270 ComG operon protein 1 2.5
lr2135 EU038264 Membrane protein, putative 1.5
lr2136 EU038265 Hypothetical protein 1.6
lr2137 EU038266 Amino acid transporter 1.6
Replication, recombination, and repair
Chromosomal replication initiator
lr0138 EU038222 protein DnaA 1.6
Nuclease subunit of the
lr0311 00857869 excinuclease complex 1.7
Ir1238 EU038240 DNA gyrase, A subunit 1.8
Ir1239 EU038241 DNA gyrase, B subunit 1.6
jignal transduction mechanisms .
' Stress response membrane
lr0279 EF421950 GTPase TypA 1.6
Transcriptional regulator, MarR
lr1012 EF421886 family 1.8
Transcription
DNA-directed RNA polymerase,
lr1358 EU038245 beta subunit 1.6
Translation, ribosomal structure and biogenesis
lr0050 EU038217 Ribosomal protein L14 2.3
lr0051 EU038218 Ribosomal protein S17 2.2
lr0062 EF421960 Ribosomal protein S8 2.1
Ir0063 EU038219 Ribosomal protein L6 2.0
lr0251 00219950 Rnase P protein component 1.7
Ir0286 EU038227 Ribosomal protein S13p 1.9
lr0287 EU038228 Ribosomal protein S11 2.0
lr0470 EF421870 Ribosomal protein L15 1.9
Ir0471 EU038230 Ribosomal protein L30 1.9
lr0472 EU038231 Ribosomal protein 35 1.9
TetW, Tetracycline resistance
lr1996 EF421935 protein 1.8

 

Table 2.10 (cont’d)

 

Substrate transport and metabolismd

 

 

lr0092

Ir0101

Ir0136

lr0137
lr0160

Ir0324

#0467
00673
k0674

h1009

Ir1013

"1198

k1287

k1417
01419
”1508
"1961
"1963
01964
01967

EF421961

EF421948

EU038221

EF537897
EF547651

EF421866

EU038229
EU038238
EU038239

EF421883

EF421887

00074887

EF421 909

D0233690
00233692
EU038250
D0857830
00857832
00857833
DQ857836

Deoxyribose-phosphate aldolase
(EC 4.1.2.4)
Phosphopentomutase (EC
5.4.2.7)

Spermidine/putrescine ABC
transporter, permease protein

Spermidine/putrescine ABC
transporter, permase protein
Sugar kinase, ribokinase family
Cystathionine beta-lyase (EC
4.4.1.8) / Cystathionine gamma-
lyase (EC 4.4.1.1)

Adenylate kinase (ATP-AMP
transphosphorylase)

Purine nucleoside
phosphorylase
Pyrimidine-nucleoside
phosphorylase
(acyl-carrier-protein) S-
malonyltransferase, FabD
3-hydroxymyristoyl/3-
hydroxydecanoyl-(acyl carrier
protein) dehydratase, FabA
HisJ ABC-type amino acid
transport/signal transduction
systems periplasmic
component/HisM ABC-type
amino acid transport system
permease component
3-oxoacyl-(acyl-carrier—protein)
synthase, FabB
Triphosphoribosyl-dephospho-
COA synthetase

Citrate lyase beta subunit
Permease, GntP family

CbiP, cobyric acid synthase
Cbi0, cobalt transport protein
CbiN, cobalt transport protein
CbiK, cobalt chelatase

81

3.1

2.9

1.6

1.8
2.3

2.1

1.8
1.7
2.9

1.6

1.8

1.9

1.7

1.9
1.8
1.7
1.7
1.6
1.5
1.8

 

Table 2.10 (cont’d)

 

CbiH, precorrin-3B C17-
lr1970 00857839 methyltransferase 1 .6
lr2131 EU038263 Transcription regulator 2.0
lr2133 EU038271 Ribose transport protein 2.8

 

 

a GenBank accesion numbers are provided.

b Genes were classified based on COG domains found in the protein
sequence through a search of the JGI Integrated Microbial Genomes
database.

c This class represents two COG categories: cell cycle control, cell division,
and chromosome partitioning and cell wall/membranelenvelope biogenesis.

d This class represents multiple COG categories that include transport and
metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids,
inorganic ions, and secondary metabolites.

Acknowledgements. We would like to thank Eamonn Connolly for helpful
discussions and access to the L. reuteri ATCC 5730 genome and Eric Hufner
and Christian Hertel for assistance in generating mutants. We also thank Jeff
Landgraf in the Research Technology Support Facility at Michigan State
University for assistance with microarray production. This work was supported in
part by funding support to RAB from the Michigan State Center for Microbial

Pathogenesis and the Rackham Foundation.

82

10.

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87

CHAPTER 3

CHARACTERIZATION OF THE BILE SALT HYDROLASE ACTIVITY OF

LACTOBA CILLUS REUTERI ATCC PTA 6475
INTRODUCTION

The gastrointestinal microbiota plays an important role in the normal circulation
and excretion of human bile. The primary bile acids, cholate and
chenodeoxycholate, are synthesized in the liver from cholesterol. Before
secretion into the small intestine, these bile acids are conjugated to either a
glycine or taurine molecule, which improves the solubility and detergent
properties of these molecules (18). As bile passes through the small intestine
into the colon, the bile acids that are not reabsorbed in the ileum are modified by
gastrointestinal bacteria. The first step in this modification is removal of the
taurine or glycine molecule through the activity of bile salt hydrolase enzymes.
This modification then allows for further alterations such as those carried out by
bacteria with 7a-dehydroxylation activity. This removal of a hydroxyl group
converts cholate and chenodeoxycholate into the secondary bile acids,

deoxycholate and lithocholate, respectively (2).

Bile salt hydrolase activity has been identified in a wide range of commensal

gastrointestinal microorganisms, including both bacteria and archaea (19), and it

88

is often suggested that bile salt hydrolase activity contributes to successful
persistence in and colonization of the host gastrointestinal tract. Tanaka et al
(27) examined 300 strains of lactic acid bacteria and found a strong correlation
between the environment from which the organisms were isolated and the
presence of bile salt hydrolase activity. The presence of bile salt hydrolase
activity in most intestinal or fecal isolates, and the absence of the activity from
strains isolated from milk or vegetable products suggests that there may be a
selective advantage in the gastrointestinal tract for strains with deconjugation
activity. Indeed, several studies have suggested that bile salt hydrolase activity
can contribute to persistence in or colonization of the gastrointestinal tract (4, 11,
13, 19). It is important to note that other studies have not demonstrated this
effect. Bateup et al (1) investigated five different strains of lactobacilli in regards
to their ability to deconjugate bile acids and colonize the murine gastrointestinal
tract. Although they demonstrated a range of bile salt hydrolase activity between
the strains (from high levels of activity to no activity), all five strains colonized
different locations in the gastrointestinal tract equally well. A separate study in
Lactobacillus johnsonii showed that even disrupting all three bile salt hydrolase
genes in this organism had no effect on gastrointestinal persistence as measured

by fecal culturing (12).

Although the activity has been primarily studied in commensal or probiotic

organisms such as Lactobacillus spp., Bifidobacterium spp., and Enterococcus

spp. (3), recent work has suggested that this activity may act as a novel virulence

89

factor for some pathogens. Research in the pathogenic Listeria monocytogenes
has revealed that the ability to deconjugate bile acids is not only controlled by
PrfA, a transcriptional regulator known to control other virulence factors, but that
the activity also plays an important role in the infection capabilities of this

pathogen (4, 13).

Despite the fact that bile salt hydrolase activity is commonly found in
gastrointestinal microorganisms and is believed to contribute to persistence of
commensals and some pathogens in the gastrointestinal tract, the actual purpose
of this activity is yet to be determined. Possibilities include: detoxification of
conjugated bile acids, contribution to gastrointestinal persistence, nutritional
benefit through utilization of the cleaved amino acid moiety, or induction of
alterations in the cell membrane that may make that bacteria more resistant to
other stresses encountered in the gastrointestinal tract such as defensins (3). An
understanding of the function of bile salt hydrolase activity in regards to the
bacteria is necessary as the activity also has important consequences on the
host. Many probiotic strains exhibit the ability to deconjugate bile acids, and
several of these strains are being investigated for the ability to lower serum
cholesterol levels in the host. It has been proposed that administration of
bacteria with bile salt hydrolase activity may lower cholesterol levels in two ways.
The first proposed mechanism is that cholesterol may co-precipitate with
deconjugated bile acids, thus causing a higher excretion of cholesterol. The

second is that administration of bacteria with this activity may increase the

90

amount of deconjugated bile acids that are excreted, thus increasing the amount
of bile acids that need to be synthesized from cholesterol (22, 31, 32). Despite
this proposed beneficial effect, various studies have demonstrated that an
excess of bacteria with bile salt hydrolase activity in the gastrointestinal tract may
lead to abnormal digestive functions, gallstone formation, and possibly a higher
tendency towards colon cancer (3). Therefore, a thorough understanding of the
role that bile salt hydrolase activity plays regarding both the bacteria and the host

is needed.

MATERIALS AND METHODS
Strains and media. The strains and vectors used in this study are listed in Table
3.1. L. reuteri strains were grown in MRS medium (Dich). PRB240 was grown

in MRS containing 10 ug/mL erythromycin. Individual bile acids, as well as ox

gall/bovine bile, were ordered from Sigma.

91

Table 3.1. Bacterial strains and plasmids used for this

 

 

 

 

 

 

 

 

 

 

study.
Reference or
Strains or plasmids Description source
Strains
Strain containing a
chromosomal copy of the
E. coli EC1000 pWV01 repA gene; Kanr (21)
Biogaia, AB,
L. reuteri ATCC 55730 Human breast milk isolate Sweden
L. reuteri ATCC PTA Biogaia, AB,
6475 Human breast milk isolate Sweden
L. reuteri ATCC PTA 6475 bile
salt hydrolase (NT01LR0487)
PRBZ40 mutant This study
Plasmids
Cmr repA-positive
temperature-sensitive
pVE6007 derivative of pWV01 (23)
Emr repA-negative derivative
pORl28 of pWV01 (21)
pORl28 + 289 bp insert from
pKW08 NT01LR0487 This study

 

 

 

 

Phenotypic plate test to determine bile salt hydrolase activity. The initial

method used to determine the presence or absence of bile salt hydrolase activity

for a particular strain was the plate method developed by Dashkevicz and

Feighner (8). For this assay, MRS agar (Difco) was combined with 0.05 to 0.5%

(w/v) of individual bile acids. The mixture was autoclaved for 20 minutes before

dividing into plates. After the plates solidified, bacterial strains were struck from

MRS plates onto the MRS plus 0.05 to 0.5% bile acid plates. Plates were

incubated anaerobically for 72 hours at 37°C. Strains were considered to be

92

 

positive for bile salt hydrolase activity based on either precipitation of the bile
acids around bacterial colonies or a change in colony morphology to white
opaque matte colonies as previously described (8). Strains were assayed for
the ability to deconjugate six conjugated bile acids: taurocholate (TCA),
glycocholate (GCA), taurodeoxycholate (TDCA), glycodeoxycholate (GDCA),

taurochenodeoxycholate (TCDCA), and glycochenodeoxycholate (GCDCA).

TLC to determine bile salt hydrolase activity. In addition to the phenotypic
plate assay, thin-layer chromatography (TLC) was used to further investigate the
ability of the strains to deconjugate the six bile acids mentioned above. For
these assays, MRS broth was combined with 1 mM of the various conjugated bile
acids listed above. The mixture was autoclaved, allowed to cool, and left in an
anaerobic chamber overnight to equilibrate before inoculation with bacterial
cultures. For inoculation, 100 pl of an overnight culture of L. reuteri grown in
MRS broth was added to 10 mL of MRS plus 1 mM bile acid. These cultures
were then incubated anaerobically at 37°C for up to 72 hours. Cultures were
assayed for bile salt hydrolase activity at either 24 or 72 hours after inoculation.

For these assays, PRB240 was grown in the presence of 10 ug/mL erythromycin.

To test for bile salt hydrolase activity, 5 mL of each culture were removed. The
mixture was acidified by adding an equal volume of 1 N HCI. Bile acids were
then extracted with 10 mL of ethyl acetate, and the phases were allowed to

separate (separation of the two phases was aided by the addition of

93

approximately one mL of a saturated brine solution). The water layer was
removed, and the organic layer was washed with 10 mL of a saturated brine
solution. After the phases separated, the organic layer (containing the bile salts)
was collected and dried over sodium sulfate. The samples were then

concentrated to dryness by rotary evaporation.

For TLC, the extracted bile acids were resuspended in approximately 0.5 mL of
methanol and spotted onto silica gel 60 F254 aluminum-backed plates, along with '
control bile acids (dilute control bile acid solutions contained purified bile acids
dissolved in methanol). The TLC plates were run using the following solvent
system: cyclohexanezethyl acetatezacetic acid (7:23:3 v/v) as previously
described (20). Using this solvent system, deoxycholate and chenodeoxycholate
migrate at the same rate. A solvent system that allowed for sufficient separation
of these two compounds was not identified. Plates were stained using 33 mM
phosphomolybdic acid dissolved in 95% ethanol and heated to visualize spots.
Cultures were considered positive for bile salt hydrolase activity based on the

appearance of spots representing deconjugated bile acids on the TLC plate.

Construction of PRBZ40 (L. reuteri ATCC PTA 6475 with disruption in
NT01LR0487). The following concentrations of antibiotics were used: 40 ug/mL
kanamycin, 10 ug/mL chloramphenicol, 10 ug/mL erythromycin (L. reuteri) or 400
ug/mL erythromycin (E. coli). The NT01LR0487 (bile salt hydrolase) mutant was

created using the system developed by Russell and Klaenhammer (26) with

94

modifications made for use in Lactobacillus reuteri (33). In short, 289 bp from
NT01LR0487 from L. reuteri ATCC PTA 6475 was cloned into pORl28. This
construct was then transformed into E. coli EC1000, and the transformed cells
were grown in LB broth in the presence of erythromycin and kanamycin. pORl28
containing the NT01LR0487 insert was then extracted and transformed into L.
reuteri ATCC PTA 6475 containing pVE6007. L. reuteri cells containing both
plasmids were grown in MRS broth containing chloramphenicol and erythromycin
at the permissive temperature of 35°C for 18 hours. The cultures were then
shifted to the non-permissive temperature of 45°C and grown in the presence of
erythromycin only. After several passages at 45°C to ensure loss of pVE6007,
individual colonies were screened for integration of pORl28 at the desired
location by PCR. Colonies were screened for the presence of both flanking
regions (using one primer that anneals to pORl28 and One primer that anneals to
the chromosome outside of the region cloned into pORl28) and the absence of a

correctly-sized wild-type gene.

Growth studies to determine the effect of loss of BSH activity. Growth
studies of L. reuteri ATCC PTA 6475 and PRB240 in the presence a synthetic
“human” bile acid mixture (SHB) were conducted. SHB was made to mimic the
bile acid concentrations in the small intestine (5, 15, 24) and contained the
following components: 0.46 mM taurocholate, 0.93 mM glycocholate, 0.46 mM

taurochenodeoxycholate, 0.93 mM glycochenodeoxycholate, 0.32 mM

95

taurodeoxycholate, and 0.64 mM glycodeoxycholate dissolved in MRS broth.
This mixture was autoclaved, cooled, and then injected into air-tight bottles

containing an anaerobic atmosphere. Overnight cultures of L. reuteri were used

to inoculate the bottles at a starting 0.0.500 = 0.02. Growth was carried out at

37° C with slow shaking. Growth curves were performed in duplicate. Samples

for TLC analysis were collected at 1, 4, 7, 10, and 23 hours of growth.

In order to investigate the effect of pH on the growth of L. reuteri ATCC PTA
6475 and PR8240 in the presence of bile, growth experiments were performed
as described above, but with the synthetic human bile mixture dissolved into a
buffered version of MRS broth. For this buffered MRS, the medium was made
from the various components: 10 g/L proteose peptone no. 3, 10 g/L beef extract,
5 g/L yeast extract, 20 g/L dextrose, 1 g/L Tween 80, 2 g/L ammonium citrate,
dibasic, 0.1 g/L magnesium sulfate, 0.05 g/L manganese sulfate, and 2 g/L
dipotassium phosphate (sodium acetate was removed). The medium was then
buffered with 200 mM MES, and the pH was adjusted to 7.0. For control
experiments, MRS without sodium acetate was made, and the pH was adjusted

to 7.0. Both types of medium were autoclaved for 20 minutes before use.

Samples for TLC analysis were collected from growth curves at 7 hours and 24
hours of growth and frozen at -20° C until use. pH was also monitored
throughout the experiments with the use of pH strips. Growth curves were

performed in triplicate.

96

RESULTS

L. reuteri ATCC PTA 6475 contains a single bile salt hydrolase that has the
ability to deconjugate the six major human bile acids. Previous work in a
variety of bacteria has demonstrated that deconjugation of bile acids is due to the
activity of one or more enzymes classified as choloylglycine hydrolases (EC
3.5.1.24). These bile salt hydrolase enzymes are responsible for the removal of
the glycine or taurine moiety from the conjugated bile acids. A search of the L.
reuteri ATCC PTA 6475 genome revealed a single gene, NT01LR0487, with
significant similarity to known bile salt hydrolase enzymes. A disruption in the
gene was created by insertion of pORl28 as described in the Materials and
Methods section. The resulting strain was titled PR8240. The ability of this
strain to deconjugate an assortment of bile acids was compared to that of the
wild-type strain, L. reuteri ATCC PTA 6475, to determine the effect of this gene

disruption.

Initial testing to determine the scope of activity for L. reuteri ATCC PTA 6475 was
performed using the plate test developed by Dashkevicz and Feighner (1989). L.
reuteri ATCC PTA 6475 was plated onto MRS plates containing between 0.05 to
0.5% taurocholate (TCA), glycocholate (GCA), taurodeoxycholate (TDCA),
glycodeoxycholate (GDCA), taurochenodeoxycholate (TCDCA), or

glycochenodeoxycholate (GCDCA) and incubated anaerobically for up to 72

97

hours. Bile salt hydrolase activity was observed by the precipitation of bile acids
surrounding individual colonies of bacteria or an opaque granular white
appearance of the colonies as shown in Figure 3.1 and as reported previously
(8). Based on these phenotypes, the plate tests revealed that the strain could

deconjugate all six of the major bile acids found in human bile.

98

'. '1

 

Figure 3.1. Different phenotypes observed using the plate test to determine
bile salt hydrolase activity. Strains of L. reuten' were plated onto MRS plates
with or without bile acids to determine bile salt hydrolase ability. A. L. reuteri
ATCC PTA 6475 and the bile salt hydrolase mutant strain, PRB240, plated onto
MRS plates containing 0.5% oxgall. Precipitation of bile is observed from ATCC
PTA 6475 as halos around the colonies; no activity is observed for PRB240.

This phenotype was also observed for plates containing glycodeoxycholate
(GDCA) and glycochenodeoxycholate (GCDCA) (data not shown). B. The two
strains (ATCC PTA 6475 and PRB240) plated onto MRS plates containing 0.01%
taurochenodeoxycholate (TCDCA). Bile salt hydrolase activity for this bile acid
was demonstrated by a cloudiness surrounding the colonies that could be
observed when the plates were held against a light. No activity is observed for
PRBZ40. C. ATCC PTA 6475 plated onto MRS or MRS plus 0.5%
taurodeoxycholate (T DCA). Deconjugation of TDCA was demonstrated by a
matte opaque granular appearance of the colonies. This phenotype was not
observed for PR8240 (data not shown).

99

Problems with the assay arose when the screen was used for testing the bile salt
hydrolase mutant strain, PRB240. This mutant demonstrated some growth
defects, particularly on the glycine-conjugated bile acids (data not shown). The
reduced growth presents a problem as the precipitation of deconjugated bile salts
is dependent on acidification of the media surrounding the bacterial colonies (8),
and this acidification depends on sufficient growth of the strain. In order to
counteract this reduced growth, lower concentrations of bile acids were used.
Under these conditions, the phenotypes for the wild-type strain were not as well
defined, particularly the matte opaque granular colony phenotype noted on plates
containing the taurine conjugated bile acids. Due to this discrepancy and to
confirm that the various phenotypes shown in Figure 3.1 did represent
deconjugation of the various bile acids, a different method, thin layer
chromatography (TLC), was used for investigating the deconjugation activity of L.

reuteri ATCC PTA 6475 and PRBZ40.

For these studies, both L. reuteri ATCC PTA 6475 and PRB240 were grown in
MRS plus 1 mM (approximately 0.05%) of each of the six major human bile acids
separately. The strains were grown for 18 hours or 72 hours and tested for
deconjugation activity by TLC. The TLC results confirmed that L. reuteri ATCC
PTA 6475 is able to deconjugate all six major human bile acids by 18 hours; no
activity was observed for PRB240, even after 72 hours of incubation (data not

shown). Thus, L. reuteri ATCC PTA 6475 contains one bile salt hydrolase gene,

100

NT01LR0487, which is able to deconjugate both the taurine and glycine-

conjugated forms of cholate, deoxycholate, and chenodeoxycholate.

Disruption of NT01LR0487 leads to a growth defect in the presence of a
synthetic human bile mixture. When L. reuteri ATCC PTA 6475 and PR8240
were grown in the presence of a synthetic human bile acid mixture (SHB), growth
defects were observed for PRBZ40. The doubling time for L. reuteri ATCC PTA
6475 in MRS broth was 39 minutes; this time slowed to 48 i 1 minutes when the
strain was grown in the presence of SHB. PRBZ40 had a doubling time of 42 i 4
minutes in the absence of bile; this time slowed to 63 i 1 minutes in the presence
of bile. Bile inhibited the growth of both strains, although the inhibition observed
for the bile-salt hydrolase mutant was more severe (a 23% increase in doubling
time was observed for the wild-type strain compared to a 50% increase for

PRB240).

Growth in the presence of bile also decreased the final culture density that the

strains were able to obtain. In the absence of bile, both strains reached a final

0.0.600 approximately equal to 4.7 after 24 hours, revealing that there is not

inherent growth defect due to the disruption of the bile salt hydrolase gene,
NT01LR0487. In the presence of bile, L. reuteri ATCC PTA 6475 reached a
culture density of 1.91 i 0.24 (40% of that obtained without bile), whereas
PR3240 reached a culture density of 0.8 i 0.01 (17% of that obtained without

bile).

101

TLC confirmed that L. reuteri ATCC PTA 6475 was able to deconjugate a mixture
of bile acids; the deconjugation products begin accumulating after 4 hours of
growth (Figure 3.2), particularly for deoxycholate and/or chenodeoxycholate. No
activity above the background level was observed for PRB240, even after 23

hours of incubation.

102

   

1. PRBZ40, 23 “Ours .\
2. MRS (n99_ °°ntro|) :19
3'6475’1hOUr ,,‘H
4. 6475, 4 hams
5. 6475, 7 “Ours
6. 6475, 23 “Ours

7. CA (pos. control)

V3

*Conjugated bile acids

V003
IVOCI

Figure 3.2. Deconjugated bile acids begin to accumulate after 4 hours in a
culture of L. reuteri ATCC PTA 6475. Representative TLC plate showing the
bile salt hydrolase activity of L. reuteri ATCC PTA 6475 against a synthetic
“human” bile acid mixture (SHB). Samples were taken a 1, 4, 7, and 23 hours.
Control solutions of cholate (CA) and deoxycholate (DCA) are included for
comparison [deoxycholate and chenodeoxycholate (CDCA) migrate at the same
rate in this solvent system]. The bile salt hydrolase mutant strain, PRB240, is
shown to have no activity at the 23-hour time point.

Controlling the pH of the growth medium alters the growth characteristics
of the wild-type and mutant strains in the presence of a synthetic human

bile mixture. Various reports in the literature have suggested that pH of the

103

environment may play an important role in the way that bacteria respond to bile
acids. Previous work in the lab has demonstrated that L. reuteri is capable of
lowering the pH of the growth medium from 6.5 down to 4.0. In order to
determine whether this shift in pH played a role in the observed growth effects
and/or deconjugation activity of L. reuteri ATCC PTA 6475 and PRB240, the cells
were grown in a buffered version of MRS broth. Results from this set of growth

curves, as well as the set described above are listed in Table 3.2.

Table 3.2. Doubling times and culture densities for L. reuteri ATCC PTA 6475
and PRB240 in the presence and absence of human bile acids.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Doubling time 0.0.500 (23-

Strain Broth Bile (mirfl 24 hours)

L. reuteri

ATCC PTA

6475 MRS - 39 4.69 1 0.01
+ 48 11 1.91 10.24

PRB240 - 42 1 4 4.71 1 0.01
+ 6311 0801001

L. reuteri

ATCC PTA MRS -

6475 acetate - 39 1 3 4.54 1 0.13
+ 47 1 3 1.52 1 0.40

PRB240 - 48 1 9 4.73 10.08
+ 66 + 11 0.70 1 0.07

L. reuteri Buffered

ATCC PTA MRS -

6475 acetate - 37 1 5 2.41 1 0.09
+ 44 1 4 2.31 10.22

PRB240 - 37 + 4 1.86 1 0.23
+ 46 1 3 2.54 1 0.12

 

104

 

Interestingly, in the buffered medium, the differences between the wild-type strain
and the bile salt hydrolase mutant regarding the lower final culture density and
the slower doubling time are no longer observed. The lack of differences
between the two strains are not due to a difference in deconjugation activity in
this medium, as confirmed by TLC (Figure 3.3). These results demonstrate that
pH does not affect the bile salt hydrolase activity of L. reuteri ATCC PTA 6475.
The growth advantage provided by bile salt hydrolase activity, however, does

appear to be pH-dependent.

105

1. MRS (neg. control)

2. Buff. MRS (neg. control)
3. PRBZ40, MRS

4. PRBZ40, Buff. MRS

5. 6475, MRS
6. 6475, Buff. MRS
7. CA (pos. control)

8. DCA (pos. control)

 

O
>

*Conjugated bile acids

V000
N00

Figure 3.3. Controlling the pH of the culture does not affect the bile salt
hydrolase activity of L. reuteri ATCC PTA‘6475. Representative TLC plate
showing the bile salt hydrolase activity of L. reuteri ATCC PTA 6475 against a
synthetic “human" bile acid mixture (SHB) in MR8 and buffered MRS. Samples
were taken at 7 hours. Control solutions of cholate (CA) and deoxycholate
(DCA) are included for comparison [deoxycholate and chenodeoxycholate
(CDCA) migrate at the same rate in this solvent system]. NO difference in activity
is observed between cultures grown in MR8 and cultures grown in buffered
MRS.

Summary of the effects of various bile acids on growth of L. reuteri.
Throughout the course of this research, various growth effects of particular bile
acids were noted for L. reuteri ATCC PTA 6475 and PRBZ40. L. reuteri ATCC

55730 was also included in several of these studies for comparison. Although

106

these experiments were carried out using different concentrations of bile acids,

and the strains were grown on MRS plates or in MRS broth, these trends

remained consistent. These qualitative observations are summarized in Table

3.3.

Table 3.3. Observed growth effects of individual bile acids in plates or in liquid
medium on L. reuteri ATCC PTA 6475, PRBZ40, and L. reuteri ATCC 55730.
Notable differences between ATCC PTA 6475 and PRB240 are highlighted in
bold. N0 = not determined. *Concentrations of greater than 0.1% were not

tested for the deconjugated bile acids due to solubility problems.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L. reuteri L. reuteri
ATCC PTA ATCC
6475 PRB240 55730
Cholate (CA)
reduced reduced
growth at growth at
0.1 % 0.1 % ND
l Glycocholate (GCA)
good growth good growth
at 0.1% at 0.1% ND
[Taurocholate (TCA)
good growth good growth
at 0.1% - at0.1% ND
Deoxycholate (DCA)
good growth good growth
at 0.1% at 0.1% ND
LGlycodeoxycholate (GDCA)
good reduced reduced
growth at growth at growth at
0.2% 0.05% 0.05%
[ Taurodeoxycholate (TDCA)
good growth good growth good growth
at 0.5% and at 0.5% and at 0.5% and
below below below

 

 

 

 

107

 

Table 3.3 (cont'd)

 

 

 

Chenodeochholate QDCA)

 

 

 

 

 

 

 

reduced reduced reduced
growth at growth at growth at
0.05% 0.05% 0.05%
Glycochenodeoxycholate
GCDCA)
severely
reduced reduced reduced
growth at growth at growth for
0.05% 0.05% 0.05%

 

 

 

 

Taurochenodeoxycholate
GCDCA)

 

 

good growth good growth good growth
at 0.5% at 0.5% at 0.5%

 

 

 

 

 

In general, these growth studies suggest that the glycine-conjugated bile acids
cause higher growth inhibition than the taurine-conjugated bile acids, and the
deconjugated bile acids cause higher growth inhibition than either of the
conjugated forms. PRBZ40, the bile salt hydrolase mutant, appears to be more
sensitive to glycodeoxycholate and glycochenodeoxycholate, suggesting that the
bile salt hydrolase activity may play a role in resistance to glyco-conjugated bile

acids.

108

DISCUSSION

The ability of bacteria to deconjugate bile acids is a trait that has been
investigated both in an attempt to understand what benefit this enzyme may
provide for the bacteria, as well as what effect the activity may have on the host.
Several large-scale studies have demonstrated a strong correlation between the
isolation of bacteria from the gastrointestinal tract and the ability of these bacteria
to deconjugate bile acids (19, 27). This correlation suggests that this activity
most likely confers some competitive advantage to strains, although the exact

function has not been elucidated.

Bile salt hydrolase activity of L. reuteri ATCC PTA 6475. These studies
revealed that L. reuteri ATCC PTA 6475 contains one bile salt hydrolase
enzyme, NT01LR0487. This enzyme is capable of deconjugating all six major
bile acids found in human bile: taurocholate (TCA), taurodeoxycholate (TDCA),
taurochenodeoxycholate (TCDCA), glycocholate (GCA), glycodeoxycholate
(GDCA), and glycochenodeoxycholate (GCDCA). PRB240, the strain containing
a disruption in NT01LR0487, was shown to have lost the ability to deconjugate
these bile acids. The substrate specificity of bile salt hydrolase enzymes
appears to be variable, as some enzymes exhibit specificity based on the steroid
nucleus of the bile acid (cholate, deoxycholate, or chenodeoxycholate) and
others exhibit specificity depending on the amino acid conjugate (taurine or

glycine). The occurrence of bile salt hydrolase enzymes capable of

109

deconjugating all six human bile acids is unknown, as many studies have
demonstrated deconjugation activities using only one or two bile acids. At least
two other studies have demonstrated bile salt hydrolase enzymes capable of

deconjugating the full range of bile acids (28, 30).

Effect of bile salt hydrolase activity during growth in the presence of bile.
Although there is a strong correlation connecting gastrointestinal strains of
bacteria with the ability to deconjugate bile acids, in vitro and in vivo data
demonstrating a direct relationship between the activity and persistence is
conflicting. One of the main proposed functions of bile salt hydrolase activity is
the detoxification of bile acids, although in vitro data regarding this is also
variable. This study demonstrated that when the pH of the growth medium was
not controlled (i.e., acidification due to metabolic products produced by the
bacteria was allowed), bile salt hydrolase activity appeared to confer an
advantage. This advantage was observed both as a decrease in doubling time
and as an increase in the final culture density achieved in the presence of bile.
This advantage was not present, however, when the pH of the medium was
controlled (not allowed to drop below 6.5). In buffered medium, the doubling
times and the final culture densities achieved by the wild-type strain and the bile-
salt hydrolase mutant were virtually identical. The pH of the culture medium in
the two situations was quite different, as in the unbuffered medium, the pH of the
culture started at 6.5 and dropped down to 3.5 in as little as six hours (depending

on the growth of the strain). In the buffered medium, the pH of the culture began

110

at 7.0 and only dropped down to 6.5, even after 24 hours of growth (data not

shown).

As most studies have demonstrated an acidic pH is necessary for optimum
activity of bile salt hydrolase enzymes (7, 17, 28, 30), and indeed one study even
observed full activity at a pH of 5.2 and a complete lack of activity at a pH of 6.8
(31 ), TLC was performed to determine what effect the varying pHs may have had
on the bile salt hydrolase activity of L. reuteri ATCC PTA 6475. The TLC data
demonstrated that there was no observable difference in the activity levels of the
enzyme in the two media types (Figure 3.3). This data strongly suggests that the
growth differences observed in the two types of media are due to an advantage

of bile salt hydrolase activity in acidic medium.

This phenomenon of differing effects of bile salt hydrolase activity at different pH
values has been reported in the literature for other bacteria, primarily in regards
to the glycine-conjugated bile acids, which are generally found to be more toxic
than the taurine-conjugated forms. De Smet et al (10) demonstrated that at a low
pH, glycodeoxycholate exhibited higher toxicity levels against strains with little to
no bile salt hydrolase activity. Grill at al (16) also demonstrated that the toxic
effect of glycodeoxycholate increased at pH values of less than 6.5 and showed
that this effect was more pronounced for strains without bile salt hydrolase
activity. Begley et al (4) also demonstrated that glycodeoxycholate exhibited

higher levels of toxicity at pH 5.5 as compared to a pH of 6.5 for Listeria

111

monocytogenes and showed that this effect was even more pronounced in a bile
salt hydrolase mutant. Interestingly, their data also demonstrated that the bile
salt hydrolase mutant had a survival advantage over the wild-type strain when
exposed to glycodeoxycholate at a pH above 5.5. These studies, along with the
experiments performed herein, suggest that there is an. advantage to bile salt
deconjugation at low pH levels. As the advantage seems to be observed
somewhere between a pH of 5.5 and 6.5, and the pH of the small intestine
ranges from 5.7 to 7.7 (25), it is difficult to surmise whether this activity

contributes a significant advantage in vivo.

Proposed model of bile salt toxicity through intracellular acidification. One
proposed function for bacterial bile salt hydrolase activity is to detoxify the
conjugated bile acids, which are proposed to inhibit cells through intracellular
acidification in the same manner as organic acids. De Smet et al (10) outlined a
model for this particular mechanism, including an explanation for the difference in
toxicity observed for taurine versus glycine-conjugated bile acids. The authors
propose that the protonated form of the conjugated bile acids is able to passively
diffuse into the cell, where it promptly becomes deprotonated due to the alkaline
intracellular environment. In a cell capable of bile salt hydrolase activity, the bile
acid is then deconjugated, producing a compound with a higher pKa (around 5.0)
whose conjugate base will pick up the extra proton before being transported
outside of the cell, thus relieving the potential intracellular acidification. In a cell

that is not capable of bile salt hydrolase activity, the cell must expend energy to

112

pump out the excess protons. The authors note that due to the lower pKa value
of taurine-conjugated bile acids (pKa of around 0.9) versus glycine-conjugated
bile acids (pKa of around 4.3), the concentration of protonated taurine-
conjugated bile acids that could passively diffuse into the cell is very low at pH
values achieved during bacterial growth. This reduced concentration is proposed

to account for the reduced toxicity observed with these bile acids.

The research presented here does not fully support this model. Although a
higher level of growth inhibition is observed for the wild-type and mutant strains
when grown in the presence of GDCA and GCDCA (compared to the taurine-
conjugated equivalents), and the defect is more severe for the mutant than the
wild-type strain, no such defect is observed when either strain is grown in the
presence of GCA (Table 3.3). As all three glycine-conjugated bile acids have
very similar pKa values, ranging from 4.27 to 4.34 (6), it is unlikely that the slight
variations in the concentration of protonated bile acids would cause this
difference in toxicity. Also, none of the taurine-conjugated bile acids exhibited an
inhibitory effect on any of the L. reuteri strains tested. Despite the fact that
taurine-conjugated bile acids are much stronger acids, and therefore the
concentration of the protonated form that is proposed to enter the cell is very low
at the pH values observed during the various culture conditions, deconjugation of
all three forms of taurine-conjugated bile acids by L. reuteri ATCC PTA 6475 was
observed. As most of the bile salt hydrolase enzymes described to date are

intracellular enzymes (3, 30), the taurine-conjugated forms must enter the cell in

113

order to become deconjugated. The lack of toxicity observed for these bile acids
against PRB240 (the mutant strain without any bile salt hydrolase activity) and for
L. reuteri ATCC 55730 (which exhibits weak bile salt hydrolase activity specific to
glycine-conjugated bile acids) suggests a different mechanism of toxicity besides

passive diffusion leading to intracellular acidification.

As many studies, including the research presented here, have demonstrated that
the deconjugated bile acids are more inhibitory to bacteria than the conjugated
bile acids (9, 14, 27, 29), the question of why bacteria would exhibit bile salt
hydrolase activity still remains to be answered. One of the current theories
specifies that the bacteria obtain some nutritional benefit from the activity,
particularly through the use of the amino acid moieties (3). Nutritional benefit for
L. reuteri appears to be an unlikely reason based on the results of this study, as
both L. reuteri ATCC PTA 6475 and PRBZ40 exhibited equal doubling times and
final culture densities during growth in the buffered medium, despite the
presence and absence of bile acid deconjugation activity. Preliminary
experiments performed to determine if the addition of taurine to the growth
medium conferred a growth advantage also suggest that bile salt hydrolase

activity does not confer a nutritional benefit (data not shown).
This research demonstrates that bile salt hydrolase activity provides a growth

advantage to L. reuteri ATCC PTA 6475 during growth at acidic pH values. The

mechanism providing this growth advantage has not yet been elucidated. The

114

lack of an effect observed in buffered MRS medium suggests that the advantage
is pH-dependent, although most of the data does not support the previous model
of prevention of intracellular acidification due to passive diffusion of the
conjugated bile acids into the cell. Further studies, including examination of the
intracellular pH values of the cells under the various growth conditions,
investigation of the potential role of a conjugated bile acid transporter, and closer r
analysis of the specific rates of deconjugation for individual bile acids may lead to
an understanding of the mechanism through which this advantage is provided to

L. reuteri ATCC PTA 6475.

 

Acknowledgements. The buffered MRS recipe was developed by Cathy
Robinson. TLC was performed in the lab of Dr. Babak Borhan, with assistance

from Daniel Whitehead.

115

10.

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119

CHAPTER 4

IDENTIFICATION OF A PUTATIVE NOVEL IMMUNOMODULATORY

COMPOUND FROM LACTOBACILLUS REUTERI

INTRODUCTION

Probiotics are live microorganisms that confer health benefits to the host when
administered in adequate amounts (6, 8). Many probiotics are now marketed to
consumers and include organisms such as lactobacilli, streptococci,
bifidobacteria, E. coli Nissle 1917, and the yeast Saccharomyces boulardii (12).
In general, probiotic lactobacilli are considered safe for human consumption
based on previous clinical trials, epidemiological studies, and historical usage in
fermented foods (1, 23). However, the mechanisms by which probiotics promote
good health and combat diseases are poorly understood. A better understanding
of how probiotics influence the health of the host is critical to utilizing these

organisms to their fullest potential.

One emerging area of probiotic research is the ability of these bacteria to alter
the immune system. Several probiotics secrete immunomodulins that modulate
the host inflammatory response, but the bacterial products responsible for the
effects on inflammation are still undefined (7, 17, 19). The inflammatory cytokine

TNF, which is produced primarily by monocytes and macrophages, is a key

120

mediator of intestinal inflammation. lmmunoprobiotics, or probiotics producing
key immunomodulatory factors, are potential therapies for various immune-
mediated disorders such as Crohn’s disease (14). Indeed, select L. reuteri
strains inhibit TNF production by monocytoid THP-1 cells and monocytes isolated
from patients with Crohn’s disease (15, 19). Strains of L. reuteri capable of
inhibiting TNF were able to reduce inflammation in a H. hepaticus-induced
murine model of inflammatory bowel disease (19). The mechanism(s) behind

this down regulation of inflammation are not understood.

Bacterial species can be identified based on their fatty acid composition. In
1950, the first cyclopropane fatty acid (CFA) was identified as cis-11,12-
methylene octadecanoic acid, or lactobacillic acid, in L. arabinosus (9).
Additional CFAs have been identified in gram-negative and gram-positive
bacteria, including dihydrosterculic acid (4). As shown in Figure 4.1, lactobacillic
and dihydrosterculic acids are produced from the precursors, vaccenic acid and
oleic acid respectively, by the enzyme cyclopropane fatty acid synthase (Cfa).
Lactobacillic and dihydrosterculic acids are 19 carbon CFAs that differ only in the
placement of the propane ring. During synthesis, Cfa adds a methylene carbon
across the double bond forming a three-carbon ring using S-adenosylmethionine
as a methyl donor. While our knowledge of how CFAs are produced has greatly
increased in the past decade, the physiological role(s) of CFAs are poorly

understood. CFAs may stabilize the cellular membrane, allowing bacteria to

121

survive exposure to adverse conditions such as extreme acid shocks and

repeated freeze/thaw cycles (4).

OH OH

Oleic Acid Dihydrosterculic Acid

V

Cyclopropane Fatty Acid Synthase

/OH—\ OH

Vaccenic Acid 0 Lactobacillic Acid

    

Figure 4.1. Synthesis of cyclopropane fatty acids. Cyclopropane fatty acid
synthase converts oleic acid and vaccenic acid into dihydrosterculic acid and
lactobacillic acid, respectively (4).

Microbial fatty acids and lipids have been previously shown to have
immunomodulatory properties. Lipid A is the active component of bacterial LPS,
3 known agonist of TLR2 and TLR4. The length and degree of saturation of the
fatty acid chains of Lipid A are important determinants of LPS activity (18).
Plasmodium falciparum produced a mixture of fatty acids that was more effective

at inhibiting TNF production than any sole fatty acid tested (5). A Mycobacterium

tuberculosis mutant unable to synthesize trans-cyclopropyl mycolic acids was

122

more virulent and stimulated murine TNF production by bone marrow-derived
macrophages and RAW 264.7 macrophages more than the wild type strain (21 ).
However, the effects of cyclopropane fatty acids produced by probiotic bacteria

on the host inflammatory responses are poorly understood.

This report demonstrates that synthesis of the specific CFA, lactobacillic acid, is
restricted to TNF-inhibitory L. reuteri strains. The production of lactobacillic acid
correlates with the emergence of the ability to inhibit TNF production. L. reuteri
strains incapable of producing lactobacillic acid have an insertion of DNA at the
3’ end of the cfa gene, which may affect their ability to synthesize lactobacillic
acid (Figure 4.7). We have constructed a L. reuten' cfa mutant and shown that it
is unable to synthesize lactobacillic acid. The cfa mutant has partially lost the
ability to inhibit TNF production in an LPS-activated macrophage model.
Possible direct and indirect effects of lactobacillic acid on immunomodulation are

discussed.
MATERIALS AND METHODS

Key reagents, bacterial strains and mammalian cell lines. All chemical
reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise
stated. Polystyrene plates were obtained from Corning (Corning, NY).
Polyvinylidene fluoride membrane filters of 0.22 pm pore size (Millipore, Bedford,

MA) were used to yield L. reuteri-derived cell-free supernatants. All bacterial

123

strains and plasmids are described in Table 4.1. L. reuteri strains were cultured
in deMan, Rogosa, Sharpe (Difco, Franklin Lakes, NJ) or LDMIIIG (supplemental
material) media unless otherwise state. PRB173 was grown in the presence of
10 ug/mL erythromycin. Cis-vaccenic acid was ordered from MP Biomedicals
(Ohio). For anaerobic culturing, an anaerobic chamber (1025 Anaerobic System,
Forma Scientific, Waltham, MA) supplied with a mixture of 10% CO2, 10% H2,
and 80% N2 was used. THP-1 cells (ATCC TlB-202) were maintained in RPMI

1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) at

37°C and 5% CO2.

Table 4.1 — Bacterial strains and plasmids used in this study.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Bacterial Strains and
Plasmids Description Source
Bacterial strains
L. reuteriATCC Isolate from Peruvian mother’s Biogaia AB
55730 milk (Raleigh, NC)
L. reuteri ATCC Isolate from Finnish mother’s Biogaia AB
PTA 6475 milk (Raleigh, NC)
L. reuteriATCC Oral isolate from Japanese Biogaia AB
PTA 5289 female (Raleigh, NC)
L. reuteri CF48-3A Fecal isolate from Peruvian Biogaia AB
child (Raleigh, NC)
L. reuteri PRB173 L. reuteri ATCC PTA 6475 This study
pPBR173 inserted into cfa
E. coli EC1000 chromosomal copy of the (13)
pWV01 repA gene; Kanr
Plasmids
pVE6007 Cmr repA-positive temperature- (16)
sensitive derivative of pWV01
pORl28 Emr repA-negative derivative of (13)
pWV01
pPRB173 pORl28 + 239 bp insert from This study
NT01LR1 143

 

 

124

 

Creation of PRB173 (L. reuteri ATCC PTA 6475 with a mutation in
NT01LR1143). The following concentrations of antibiotics were used: 40 ug/mL
kanamycin, 10 ug/mL chloramphenicol, 10 ug/mL erythromycin (L. reuten) or 400
ug/mL erythromycin (E. coli). The NT01LR1143 (cyclopropyl fatty acid synthase)
mutant was created using the system developed by Russell and Klaenhammer
(22) with modifications made for use in Lactobacillus reuteri (26). In short, 239 bp
from NT01LR1143 from L. reuteri ATCC PTA 6475 was cloned into pORl28.
This construct was then transformed into E. coli EC1000, and the transformed
cells were grown in LB broth in the presence of erythromycin and kanamycin.
pORl28 containing the NT01LR1143 insert was then extracted and transformed
into L. reuteri ATCC PTA 6475 containing pVE6007. L. reuten' cells containing
both plasmids were grown in MRS broth containing chloramphenicol and
erythromycin at the permissive temperature of 35°C for 18 hours. The cultures
were then shifted to the non-permissive temperature of 45°C and grown in the
presence of erythromycin only. After several passages at 45°C to ensure loss of
pVE6007, individual colonies were screened for integration of pORl28 at the
desired location by PCR. Colonies were screened for the presence of both
flanking regions (using one primer that anneals to pORl28 and one primer that
anneals to the chromosome outside of the region cloned into pORl28) and the

absence of a correctly-sized wild-type gene.

125

Samples for fatty acid methyl ester (FAME) analysis of overnight cultures
(strain comparison and mutant analysis). Cultures for analysis were grown
aerobically without shaking for 18 hours in 10 mL of MRS (Difco) at 37°C (L.
reuteri CF48-3A was grown at 35°C by S. E. Jones). PRB173 was grown in the
presence of 10 ug/mL erythromycin. Cultures were spun down at 4000 rpm for 10

minutes at room temperature. Supernatant was removed. Pellets were

 

r
resuspended in one ml of fresh MRS and spun at 13,400 rpm for 2 minutes. I
Supernatant was removed and pellets were frozen at -80°C until analysis. i
Experiments for strain comparisons were performed in quadruplicate. For mutant

versus wild-type comparison, six wild-type samples were analyzed and four Ii

mutant samples were analyzed.

Samples for FAME analysis at various stages of growth. Cultures of L.
reuten' ATCC PTA 6475 for analysis were grown in the presence of 2% oxygen
with slow shaking in 110 mL of MRS (Difco) at 37°C. Culture density was
measured at an optical density (0.0.) of 600 nm. 50 mL samples were collected
from the cultures at mid-log (0.0. = 0.4); 10 mL samples were collected from the
cultures at early stationary (0.0. = 3.1), late stationary (0.0. = 4.2), and after
overnight incubation (approximately 18 hours). A representative growth curve is
shown in Figure 4.2. After collection, samples were processed as described
above, and pellets were frozen at -80°C until analysis. Experiments were

performed in triplicate.

126

 

I

10

0.0.600

0.1 .

 

 

0.01 . T . fl
0 200 400 600 800

time (min)

 

 

 

Flgure 4.2. A representative growth curve for L. reuten’ ATCC PTA 6475.
Samples for fatty acid analysis (mid-log, early stationary, and late stationary
phase) were collected at the time points represented by open circles.

Samples for FAME analysis In dlfferent variations of MRS medla. Three
variations of MRS media were used to determine the effects of various media
components on the fatty acid composition of L. reuten‘ ATCC PTA 6475 and
PRB173. Cultures were grown aerobically in 10 mL of MRS or modified MRS for
18 hours without shaking at 37°C. PRB173 was grown in the presence of 10
pg/mL erythromycin. Complete MRS (MRS A to distinguish it from the Difco
MRS) was made from the following components: 10 g/L proteose peptone no. 3,
10 g/L beef extract, 5 g/L yeast extract, 20 g/L dextrose, 1 g/L Tween 80, 2 g/L
ammonium citrate, dibasic, 5 g/L sodium acetate, 0.1 g/L magnesium sulfate,
0.05 g/L manganese sulfate, and 2 g/L dipotassium phosphate. Two variations

of MRS media were also made. The first, MRS B, is MRS media made without

127

 

beef extract or Tween 80. The second, MRS C, is MRS media made without
beef extract or Tween 80 with the addition of 0.01% cis-vaccenic acid (w/v).
Samples were processed as listed above, and pellets were frozen at -80°C until

analysis. Experiments were performed in triplicate.

Fatty acid analysis. All fatty acid analysis was performed by Microbial lD

 

 

Ir-
(Newark, DE; mpzllwwwmicropjalidcom).

Preparation of cell-free supernatants for immunomodulation studies. For '_
planktonic cells, 10 mL of LDMIIIG was inoculated at a starting 00600 = 0.1 (~7 x i

107 CF U) using 16-18 hr L. reuteri cultures. Bacteria were incubated for 24 hr at
35°C in anaerobic conditions. Serial dilutions were plated onto MRS medium to
determine cell counts. Cells were pelleted (4000 x 9, RT, 10 minutes) and
discarded. Supernatants were filter-sterilized. Aliquots were vacuum-dried and
re-suspended to the original volume using RPMI. For biofilms, L. reuteri cultures
(16-18 hrs of incubation) were diluted 1:100 in MRS and aliquots were placed
into polystyrene 24-well plates. Plates were incubated anaerobically for 24 hr at
35°C. Supernatants and planktonic cells were removed by aspiration, and
biofilms were washed with 50 mM sodium phosphate buffer (37°C, 100 rpm, 10
minutes). One mL of LDMIIIG was added to each well, and the plates were
incubated for 2 hours at 35°C in anaerobic conditions. The supernatants were

filtered through 0.22 gm filters, vacuum-dried and resuspended in RPMI to the

128

starting volume. Biofilms were removed by sonication (5 min, 20°C), and serial

dilutions were plated to determine cell counts.

TNF inhibition experiments. TNF assays were previously described (15).
Briefly, cell-free supernatants from L. reuteri planktonic or biofilm cultures (5%

v/v) or serial dilutions of lactobacillic acid (2% v/v) were added to human THP-1

 

cells (approximately 5 x 104 cells) activated with E. coli O127:B8 LPS (100 T
ng/mL). After the addition of L. reuteri supernatants and LPS, plates were

incubated at 37°C with 5% CO2 for 3.5 hours. Trypan blue (Invitrogen, Carlsbad,

CA) was employed to ascertain cell viability. THP-1 cells were pelleted (1500 x i

g, 5 min, 4°C), and quantitative ELISAs (R80 Systems, Minneapolis, MN) were

used to determine the amount of TNF present in monocytoid cell supernatants.

Statistical analyses for TNF bioassays and fatty acid profiles. For the TNF
bioassays, a minimum of three biological replicates were performed and
analyzed by ANOVA. Differences were considered statistically significant if p
<0.05. Fatty acid profiles were determined a minimum of three times. All error

bars in the figures represent standard deviations.

RESULTS

lmmunomodulatory activity of TNF inhibitory L. reuteri strains is produced

upon entry into stationary phase. Some probiotic L. reuteri strains have the

129

ability to produce secreted factors that suppress lipopolysaccharide (LPS)
induced TNF production in primary monocytes and macrophages (15). This
activity is strain specific. We have identified two strains of L. reuteri (ATCC PTA
6475 and ATCC PTA 5289) that secrete one or more compounds that exhibit
potent immunomodulatory activity against TNF production in activated
macrophages. Secreted compounds from a third strain included in this study, L.
reuteri ATCC PTA 4659, have also been shown to inhibit TNF production (3). T
Two additional strains (L. reuteri ATCC 55730 and CF-48/3A) were incapable of

reducing TNF production (11). The inhibitory effect on TNF production is found

 

in culture supernatants of cells grown into stationary phase and is absent from t
culture supernatants of exponentially growing L. reuteri. The goal of this study
was to identify the immunomodulatory compounds produced upon entry into

stationary phase using a combined genomics and genetic approach.

The cyclopropyl fatty acid, lactobacillic acid, is specifically produced in
immunomodulatory strains of L. reuteri. Fatty acids have been shown to play
important roles in the regulation of the immune system. To identify potential
membrane fatty acids that are produced specifically by immunomodulatory L.
reuteri strains we compared the membrane fatty acid profiles of the three strains
capable of inhibiting TNF production with the two strains that are incapable of
downregulating TNF production. These five strains were grown to stationary
phase and fatty acid methyl ester analysis was performed to determine the

membrane fatty acid content. The results for the major fatty acids are shown in

130

Figure 4.3. Briefly, the immunomodulatory strains appeared to be very similar to
one another; they consisted of approximately 41% palmitic acid (16:00), 11%
Oleic acid (18:1 w9c), 11% vaccenic acid (18:1 (97c), 5% stearic acid (18:00),
18% dihydrosterculic acid (19:0 cyclo w9c), and 10% lactobacillic acid (19:0 cyclo
(98c). The immunoneutral strains, L. reuteri ATCC 55730 and L. reuteri CF48-

3A, consisted of approximately 26% palmitic acid, 21% oleic acid, 17% vaccenic

 

_-
acid, 4% stearic acid, and 24% dihydrosterculic acid. Interestingly, the
immunomodulatory strains contained a novel cyclopropyl fatty acid, lactobacillic L
acid (phytomonic acid) that was absent from the immunoneutral strains. Thus .1
I
t
I

lactobacillic acid is a possible candidate immunomodulin produced by L. reuteri
strains ATCC PTA 6475, ATCC PTA 5289, and ATCC PTA 4659. A second
cyclopropyl fatty acid, dihydrosterculic acid, was present in both immunoneutral
and immunomodulatory strains in similar amounts. This indicated that
immunoneutral strains were capable of producing fatty acids containing

cyclopropane rings.

131

I Palmitic '3 Oleic Vaccenic l Stearic D Dihydrosterculic U Lactobacillic
45

 

40

35*

30

25

Percentage of total fatty acids

 

     

   

    

 

 

0 l, ”.4 ., ........_. . 1.1. .. ..- _. .... ._ .._..... L. “2.... LL .
ATCC 55730 CF48-3A ATCC PTA ATCC PTA ATCC PTA
5289 4659 6475

Figure 4.3 - TNF-inhibitory strains produce lactobacillic acid. Cultures of
five strains of L. reuteri were grown in MRS broth for 18 hours at 37° C. Fatty
acid profiles were determined by gas chromatography. ATCC 55730 and CF48-
3A (TNF non-inhibitory strains) do not produce the CFA, lactobacillic acid. In
contrast TNF-inhibitory strains (ATCC PTA 6475, ATCC PTA 5289, and ATCC
PTA 4659) produce lactobacillic acid. Experiments were performed in triplicate;
error bars represent standard deviation. * Lactobacillic acid.

The appearance of lactobacillic acid correlates with the appearance of the
immunomodulatory activity. Previous work has demonstrated that the TNF
inhibitory activity is only found in cell-free supernatant from L. reuten' cultures that
have entered stationary phase. In order to investigate whether there was a

correlation between the presence of lactobacillic acid in the cell membranes and

the presence of the immunomodulatory compound in the cell-free supernatants,

132

fatty acid profiles were compared from L. reuteri ATCC PTA 6475 cultures at
various stages of growth. According to the representative growth curve in Figure
4.2, samples for fatty acid analysis were collected from cultures at mid-log phase
(0.0.600 = 0.4), early stationary phase (0.0.500 = 3.0), and late stationary phase
(0.0.600 = 4.1). Samples were also collected after overnight incubation of
cultures (approximately 18 hours). Lactobacillic acid was not present in cultures
in mid-log phase or early stationary phases of growth (Figure 4.4). Cultures at
late stationary phase contained approximately 9.6% lactobacillic acid; overnight
cultures contained approximately 10.3%. Dihydrosterculic acid, the other
cyclopropyl fatty acid present in cultures of L. reuteri, was found at
concentrations of approximately 20% for all four time points tested (Figure 4.4).
Thus, the presence of lactobacillic acid in cultures of L. reuteri ATCC PTA 6475
correlates with the appearance of the TNF inhibitory compound(s) in cell-free

supernatant from the immunomodulatory strains.

133

 

lpalmitic loleic Dvaccenic Ddihydrosterculic Elactobacillic

 

 

Percentage of fatty acids

*

Ir- ‘

Mid-log Early stationary Late stationary Overnight

 

 

 

 

 

 

 

 

 

Figure 4.4. Lactobacillic acid appears in late stationary phase cultures of L.
reuteri ATCC PTA 6475. Cultures of L. reuteri ATCC PTA 6475 were grown in
MRS broth at 37°C. Samples were collected for FAME analysis at different
stages of growth. Experiments were performed in triplicate; error bars represent
standard deviation. *Lactobacillic acid.

The production of lactobacillic acid is due to the activity of NT01LR1143, a
cyclopropyl fatty acid synthase. Previous work in E. coli has shown that the
biosynthesis of bacterial cyclopropyl fatty acids is due to one enzyme,
cyclopropyl fatty acid synthase. This cyclopropyl fatty acid synthase (Cfa) is
responsible for the conversion of oleic acid into dihydrosterculic acid and the
conversion of vaccenic acid into lactobacillic acid (Figure 4.1). A search of the L.

reuteri ATCC PTA 6475 genome revealed a single gene, NT01LR1143, with

significant similarity to known cyclopropyl fatty acid synthases. A disruption in

134

the gene was created by insertion of pORl28 as described in the Materials and
Methods section. The resulting strain was titled PRB173. In order to confirm the
effect of the disruption of NT01LR1143, cultures of L. reuteri ATCC PTA 6475
and PRBI73 were grown overnight in MRS or MRS plus 10 ug/mL erythromycin.
Samples were collected, and fatty acid analysis was performed. The results,

shown in Figure 4.5, confirmed that the mutant strain, PRB173, was devoid of

 

I...
lactobacillic acid and contained a minor amount of dihydrosterculic acid (1.6% 9
compared to 17.8% in wild-type). The mutant strain also contained a higher j)
amount of oleic acid (52.5%) compared to the wild-type strain (10.7%). ;
:a?‘ ,
it

135

 

I ATCC PTA 6475 PRB173

 

 

 

 

 

.8601
850— '1‘
540
E
930 n-
u— I
320
O)
310
C
L3
g 0
Ir

 

 

 

 

Figure 4.5. Disruption of NT01LR1143 (CFA synthase) eliminates
production of lactobacillic acid by L. reuteri ATCC PTA 6475. L. reuteri
ATCC PTA 6475 and PRB173 were grown overnight in MRS broth at 37° C.
Samples were collected for fatty acid analysis. Experiments were performed in
triplicate; error bars represent standard deviation.

Removal of Tween 80 from the growth medium Increases the amount of
lactobacillic acid found In L. reuteri ATCC PTA 6475. By disrupting the
cyclopropyl fatty acid synthase gene (NT01LR1 143) of L. reuteri ATCC PTA
6475, a strain was created that does not contain lactobacillic acid (PRB173).
This strain will serve as an important control for future experiments directed at

determining the possible role of lactobacillic acid in suppression of TNF by

stimulated macrophages. A strain with an increased concentration of lactobacillic

136

acid would also be useful for these studies. To this aim, attempts were made to
increase the percentage of lactobacillic acid in L. reuteri ATCC PTA 6475

cultures through alteration of specific media components.

L. reuteri cultures are commonly grown in MRS broth, a rich undefined media, in
the laboratory. One component of MRS broth is Tween 80, which contains
approximately 70% Oleic acid with a balance of palmitic acid, stearic acid, and
linoleic acid. Dihydrosterculic acid, the only other cyclopropyl fatty acid present
in cultures of L. reuteri is made from oleic acid, and previous work in other
lactobacilli has demonstrated that removal of oleic acid from the medium can
increase the production of lactobacillic acid (10). In addition, the effect of adding
0.01% cis-vaccenic acid (the precursor for lactobacillic acid) in the absence of
Tween 80 was also determined. L. reuteri ATCC PTA 6475 was grown in one of
three variations of MRS broth for 18 hours, and samples were collected for fatty
acid analysis. The three variations of broth used were: MRS A (complete MRS
made from the various components), MRS B (MRS media without Tween 80 or
beef extract), and MRS C (MRS B plus 0.01% cis-vaccenic acid). A summary of
the results is shown in Figure 4.6. Briefly, in the absence of Tween 80 (MRS B),
the lactobacillic acid concentration increased from approximately 8.7% of the
total fatty acids to 29.9%, whereas the amount of dihydrosterculic acid decreased
from 15.9% to 0% in the absence of its precursor. The addition of cis-vaccenic
acid (MRS C) lead to a small increase in the lactobacillic acid, making it a total of

33.5% of the fatty acids found in the samples. Thus, growth of L. reuteri ATCC

137

 

PTA 6475 in either MRS B or MRS C would provide another sample for future
studies aimed at investigating the role of lactobacillic acid in immunomodulation.
PRB173, the CFA synthase mutant, was confirmed to be completely devoid of

lactobacillic acid in MRS B (data not shown).

138

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IMRSA IMRSB DMRsc
50
45-
40-
g
.6 35—
(0
B30—
.12
To -
E 25 ‘1'
“6 _
a, 20
9
E15—
9
8310‘
5_
04 .
'0
“s
<9
Q‘b

 

 

 

 

Flgure 4.6. Lactobacillic acld production by L. reuteri ATCC PTA 6475 can
be increased by removal of Tween 80 from the growth medium. Overnight
cultures of L. reuteri ATCC PTA 6475 were grown in MRS A (complete MRS
broth), MRS B (MRS broth with Tween 80 and beef extract removed), or MRS 0
(MRS B plus 0.01% cis-vaccenic acid); samples were collected for fatty acid
analysis. Experiments were performed in triplicate; error bars represent standard
deviation.

139

 

 

 

The cyclopropyl fatty acid synthase gene is truncated in strains of L.
reuteri incapable of down-regulating TNF production. Membrane
cyclopropane fatty acids (CFA) have been shown to increase as cells enter
stationary phase. CFA formation is performed by the enzyme cyclopropane fatty
acid synthase that is encoded by the cfa gene. We therefore scanned the
genomes of L. reuteri PTA ATCC 6475 and ATCC 55730 and identified a single
cfa gene in each genome. Interestingly, the reading frame of the cfa gene was
truncated at the 3’ end by the insertion of a DNA element in the L. reuteri ATCC
55730 but not the ATCC PTA 6475 strains (Figure 4.7). The insertion in L.
reuten' ATCC 55730 results in a C-terminal deletion of 12 amino acids in the Cfa
protein. To determine if this insertion correlated with the loss of
immunomodulatory activity we PCR amplified the cfa gene and flanking
sequences. The insertion was identified in both immunoneutral strains tested but
was absent in both strains capable of suppressing TNF. Thus the absence of the

insertion correlated with the ability to reduce TNF production.

140

 

 

ATCC PTA 6475 and ATCC PTA 5289

 

 

 

 

LR2652 RibBA "_‘ HP

 

 

 

 

 

 

 

 

 

l

Insertion

Figure 4.7. An insertion in the CFA synthase gene of L. reuteri ATCC 55730
and CF48-3A may prevent production of lactobacillic acid. PCR analysis
revealed an insertion in the region of the genome containing the cfa gene for
both immunomodulatory strains. This insertion results in the truncation of the cfa
gene. Small arrows represent the primers used for amplification of the region
surrounding the cfa gene.

Conditioned medium from a mutant defective in cyclopropyl fatty acid
production is impaired in inhibiting TNF production. To address what
possible role lactobacillic acid production has on the ability to inhibit TNF
production we compared the effects of conditioned media from wild-type cells
and the cfa mutant (PRB173) on the ability of activated monocytes to produce

TNF. Human derived THP-1 monocytes were activated by the addition of LPS

and the production of TNF was monitored by quantitative ELISA. Cell-free

141

supernatants from L. reuteri deficient in cyclopropane fatty acid synthase
(PBR173) partially lost the ability to suppress TNF when compared to the
immunomodulatory effects of wild type ATCC PTA 6475 in both planktonic

cultures and biofilms (Figure 4.8).

For planktonic cultures, TNF production was efficiently suppressed by cell-free

 

F”
supernatant derived from L. reuteri ATCC PTA 6475 compared to the LDMIIIG '_
medium control. The cfa mutant stain PRB173 was deficient in its ability to I!
reduce TNF production compared to the wild-type strain. TNF production was
approximately 2-fold higher in bioassays with PBR173 compared to bioassays E’

with wild type L. reuteri ATCC PTA 6475 (239 pg/mL and 134 pg/mL,
respectively) (p<0.001) (Figure 4.8 A). In addition, serial dilutions of probiotic-
derived supernatants were tested. At a dilution of 1:20, wild-type supernatants
still inhibited TNF by 45% compared to the media control. In contrast, at a 1:20
dilution, PRB173 supernatants had completely lost their TNF-inhibitory ability
(data not shown). We also analyzed the production of immunomodulatory
activity from biofilms and found similar trends with respect to TNF modulation.
When supernatants from biofilm cultures were tested, ATCC PTA 6475 and
PRB173 inhibited TNF production by 60% and 21%, respectively, compared to
the medium alone (Figure 4.8 B). In the presence of supernatants derived from
the Cfa-deficient strain, the quantities of human TNF produced by LPS-activated
THP-1 cells were approximately 2-fold higher than quantities observed in the wild

type strain (560 pg/mL and 282 pg/mL, respectively) (p < 0.01). Planktonic and

142

biofilm cultures of the immunoneutral L. reuteri ATCC 55730 did not suppress
TNF when compared to the media control (Fig. 4.8). In addition, mutations in
NT01LR0396 (multi-drug resistance transporter in the major facilitator
superfamily) and NT01LR1106 (putative esterase) had no effect on the ability of
L. reuteri ATCC PTA 6475 to suppress TNF, demonstrating the mutagenesis
system had no effect (data not shown). These results demonstrate that mutants
defective in lactobacillic acid production are unable to fully suppress TNF

suppression.

 

143

 

1 200

1000

800

TNF (pg/mL)
O)
O
O

.b
O
O

200

 

 

 

Media

 

D < 0.001

   

ATCC PTA 6475

 

 

 

   

PBR173 ATCC 55730 '

 

 

 

B
1 000

800

O)
O
O

TNF (pg/mL)
A
O
O

200

 

 

 

Media

D < 0.01

 

 

ATCC PTA 6475

 

 

 

 

PBR173 ATCC 55730

 

144

Figure 4.8 - L. reuteri deficient in Cfa had diminished ability to suppress
TNF. Cell-free supernatants from L. reuteri cultured as planktonic cells or
biofilms were tested for the ability to inhibit LPS-activated THP-1 cells from
producing TNF. When cultured as planktonic cells (A) or biofilms (B), PBR173,
which does not produce lactobacillic acid, had diminished ability to suppress TNF
production compared to wild-type cells.

DISCUSSION

The ability of probiotics to affect the immune system of the host is likely to be an
important mechanism for how these types of bacteria benefit human health.
Several gastrointestinal disorders, including Crohn’s disease, are partly caused
by increased inflammation in the intestine, and therapies that target pro- D}

inflammatory cytokines such as TNF have been effective in providing

 

symptomatic relief is some cases. Thus candidate probiotics that can
downregulate pro-inflammatory cytokines in the intestinal tract offer a means of y
localized delivery of an immunomodulatory agent. Several studies have

identified potent immunomodulatory activities expressed by candidate probiotic

bacteria, however in most cases the bacterial factors mediating these activities

are not understood.

In this study we have identified the cyclopropane fatty acid lactobacillic acid as a
candidate immunomodulin involved in the inhibition of TNF produced by activated
macrophages. Several lines of evidence support that L. reuteri strains that
produce lactobacillic acid have increased capabilities of inhibiting TNF. First, the
presence of lactobacillic acid was only found in strains capable of down-
regulating TNF production. Second, the appearance of lactobacillic acid in
stationary phase correlates with the appearance of immunomodulatory activity.

Third, mutation of cfa, which eliminates the production of lactobacillic acid,

145

results in a L. reuteri strain with reduced capacity to inhibit TNF production from
both planktonic cultures and biofilms. These results indicate that lactobacillic
acid is either an immunomodulatory compound itself or loss of lactobacillic acid

indirectly effects the production of another immunomodulin.

We have had lactobacillic acid synthesized and have not detected TNF inhibition
by lactobacillic acid at non-cytotoxic concentrations (data not shown). This either
indicates that lactobacillic acid does not directly play a role in suppression of TNF
expression or that we have not identified the proper conditions for delivering
lactobacillic acid to macrophages. This could be due to either biological or
technical reasons. We found that lactobacillic acid was very insoluble as a free
fatty acid and therefore it is unclear that we were successful in delivering
lactobacillic acid properly to the macrophages. In other situations fatty acids
delivered to cells in phospholipids are able to mediate effects not observed with
free fatty acids (7). We are currently attempting to use petroleum ether extracted
lipids from the immunomodulatory L. reuteri strains to determine if we can

reconstitute immunomodulatory activity.

The effects of cyclopropanation in lipids on the immune response has recently
been studied in Mycobacterium tuberculosis. The disruption of cyclopropyl ring
formation in mycolic acids has a profound effect on how this pathogen interacts
with the immune system. Deletion of the cmaZ gene, which encodes a

cyclopropane synthase enzyme which adds trans-cyclopropyl rings to mycolic

146

II. [Ila-"'2. “Ill

 

. .1 .- '31;

«.31

 

acids, produces a hypervirulent strain in a mouse model of tuberculosis.
Induction of pro-inflammatory cytokines in murine-derived macrophages was
more pronounced with the mutant strain and this could be mimicked with phenyl
ether extracted lipids. This supports the claim that trans-cyclopropanation of
mycolic acids attenuates the host response to M. tuberculosis (21). Interestingly,
deletion of the pcaA gene, which encodes another cyclopropyl mycolic acid
synthase in M. tuberculosis and is responsible for the synthesis of cis-cyclopropyl
rings in mycolic acids, had the opposite effect on virulence and in vitro
inflammatory responses (20). This work indicates that cyclopropane ring fatty
acids can influence pathogenicity and the host immune response. Whether or

not lactobacillic acid has a similar direct effect remains to be determined.

Although we currently favor a direct effect of lactobacillic acid on the
inflammatory response of LPS-stimulated macrophages, it is equally possible
that the observed effects of deletion of Cfa are indirect. We have identified 36
genes that are significantly reduced in expression when cfa is disrupted, each of
these are candidates for being involved in immunomodulation (Dephine Saulnier
and Jim Versalovic, personal communication). The most interesting gene from
this group is a putative cyclase(NT01LR1833) that is found only in some strains
of L. reuteri but not other lactobacilli. Interestingly, L. reuteri ATCC 55730 does
not contain this gene, again correlating the absence of this gene with a lack of
immunomodulatory activity. The function of this cyclase is unknown.

NT01LR1833 is located in a putative operon with a cobalamin-independent

147

 

 

 

 

methionine synthase and near an enzyme that catalyzes the final step in
menaquinone (vitamin K) biosynthesis. Vitamin K, which is an essential vitamin
for humans, has been linked to the regulation of the immune response. We are
currently investigating whether or not this putative cyclase has any role in

immunomodulation.

Further work is needed to determine if lactobacillic acid plays a direct or indirect
role in modulation of the host immune response. The creation of a mutant strain
of L. reuteri ATCC PTA 6475, PRB173, that is incapable of producing
lactobacillic acid, as well as the identification of growth conditions that triple the

lactobacillic acid content of the cell membranes will aid in these pursuits.

Acknowledgements. This project was carried out in collaboration with
researchers at Baylor College of Medicine. Sara E. Jones grew the overnight
cultures of L. reuteri CF48-3A (a second immunoneutral strain) for fatty acid
analysis and performed the TNF inhibition experiments (including growth of
bacterial and THP—1 cells, preparation of cell-free conditioned medium, and TNF
assays). Delphine Saulnier identified‘the truncation in the cfa synthase gene of
the immunoneutral L. reuteri strains. The author would also like to acknowledge

Daniel Whitehead for production of Figure 4.1.

148

 

 

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Law, J., G. Buist, A. Haandrikman, J. Kok, G. Venema, and K.
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Lin, Y., and J. Versalovic. 2006. lmmunoregulation by probiotics.
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Lin, Y. P., C. H. Thibodeaux, J. A. Pefia, G. D. Ferry, and J. Versalovic.
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Maguin, E., P. Duwat, T. Hege, D. Ehrlich, and A. Gruss. 1992. New
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Menard, S., C. Candalh, J. C. Bambou, K. Terpend, N. Cerf-

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Netea, M. G., M. van Deuren, B. J. Kullberg, J. M. Cavaillon, and J. W.
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Pefia, J. A., A. B. Rogers, Z. Ge, V. Ng, S. Y. Li, J. G. Fox, and J.
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Appl. Environ. Microbiol. 73:3924-3935.

Walter, J., P. Chagnaud, G. W. Tannock, D. M. Loach, F. Dal Bello, H.

F. Jenkinson, W. P. Hammes, and C. Hertel. 2005. A high-molecular-
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151

 

 

CHAPTER 5

SUMMARY AND SIGNIFICANCE

Although research in the field of probiotics has increased exponentially over the
past twenty years, the mechanisms by which these organisms exert a beneficial
effect on the host are not well understood. Much research has focused on in
vitro studies investigating survival of stresses that the bacteria will be exposed to
in the gastrointestinal tract, the ability to inhibit pathogens, and the ability to
modulate the host immune response. In vivo studies are investigating a wide
range of disorders including bacterial infections, allergies, and gastrointestinal
disorders. Although various strains of L. reuteri have demonstrated effectiveness
in various in vitro and in vivo studies as discussed in Chapter 1, the mechanisms
by which this effectiveness is achieved has remained elusive. For example,
there appears to be wide ranging differences between the physiology and effects
of particular strains within the species, and very little has been determined

regarding the actual interactions between L. reuteri and the host.

Strain specificities. Differences between strains of L. reuteri need to be
thoroughly investigated. Initial studies have demonstrated that there are
differences in the stress resistance, production of antimicrobial compounds,
immune response modulation, and effectiveness in animal trials. For example,

one of the common characteristics of L. reuten' that is often attributed for the

152

 

 

 

ability of the species to cause beneficial effects is the production of the wide
spectrum antimicrobial compound, reuterin. Research has shown that at least
one strain, L. reuteri RC-14 does not produce reuterin, nor does it contain the
necessary enzyme needed to convert glycerol into reuterin (4). Thus, any
probiotic effects observed with this strain are not due to reuterin production,
although this observation does not rule out reuterin’s importance for other

beneficial effects.

These investigations have identified some of the factors involved in the survival
of L. reuteri in the host during exposure to bile in the small intestine, as well
identified a putative novel immunomodulatory compound, lactobacillic acid.
These studies have also highlighted some of the strain specificities that may play
important roles in interactions with the host. Althoughall L. reuteri strains
included in this work were of human origin, differences were observed in the
mechanisms used for survival in the presence of bile. L. reuteri ATCC PTA 6475
appears to obtain a benefit from bile salt hydrolase activity during growth in the
presence of bile. Preliminary research suggests that this strain’s bile salt
hydrolase enzyme is active against a wider range of bile acids and acts more
quickly than the enzyme from L. reuteri ATCC 55730. Interestingly, several of
the genes identified as playing an important role in the survival and growth of L.
reuten' ATCC 55730 in the presence of bile, the putative esterase (Lr1516) and
the multidrug resistance protein (Lr1584), did not appear to have overlapping

roles in L. reuteri ATCC PTA 6475 (data not shown). Whether or not this

153

 

 

difference is due to the bile salt hydrolase activity of L. reuteri ATCC PTA 6475

has yet to be determined.

Another important strain difference is represented by the ability of some of the
strains to suppress TNF, while others do not have an effect on TNF production
by macrophages as described in Chapter 4. This difference suggests that some
strains of L. reuten' may be effective in suppressing host inflammatory responses,
while other strains may have no effect or may even exacerbate inflammation.

Fatty acid analysis of these strains revealed that all five strains investigated had

 

the ability to synthesize cyclopropyl fatty acids (all strains produced
dihydrosterculic acid, a 19-carbon cyclopropyl fatty acid); however, only the
immunomodulatory strains contained a second 19-carbon cyclopropyl fatty acid,
lactobacillic acid. Although the prevalence of lactobacillic acid among the various
strains, as well as the occurrence of lactobacillic acid at particular stages of
growth, suggest a role for the compound in TNF suppression, it is possible that

other strain differences are responsible.

Bacterial-host interactions. While strain specificity is important to investigate,
ultimately the main area lacking in probiotic research, despite much effort from
the field, is an understanding of the mechanisms through which these
microorganisms, like L. reuteri, are able to cause their beneficial effects. It is
unlikely that one common mechanism of action will be identified, thus careful
research investigating cautiously chosen strains in a range of host genetic

backgrounds should be carried out. If one strain does not alleviate a particular

154

 

disease, another strain, with different traits, should be considered. Unfortunately,
the situation could be even more complicated, as at least one study involving L.
reuteri has demonstrated that the health status of the host can alter the response
to probiotic administration (3). Ouwehand et al (9) also demonstrated that the
health status of the host tissue could alter adherence levels of probiotic bacteria.
L. reuteri ING1 exhibited better adherence to colonic tissue isolated from IBD
patients than healthy patients, as well as better adherence to mucus from
diseased tissue versus healthy tissue. Things such as inflammatory markers,

other bacteria, or mucus composition could alter this interaction (9).

One area to investigate potentially novel mechanisms of action or bacterial-host
interactions would be genes that are up-regulated during stresses found in the
host gastrointestinal system that do not appear to play a role in bacterial survival
of this stress. The putative matrix metalloprotease discussed in Appendix C is an
example of such a gene. Microarray analysis revealed that Ir1291 was up-
regulated in L. reuteri ATCC 55730 during bile shock exposure, yet mutational
analysis revealed that this gene does not appear to play a role in either survival
or growth in the presence of bile. As bile is known to act as a signal for some
pathogens to up-regulate virulence factors (5), it is possible that it may also act
as a signal for commensals or probiotic strains to up-regulate factors involved in
interaction with the host. The sequence similarity between lr1291 and eukaryotic

matrix metalloproteases, along with the important roles of matrix

155

 

metalloproteases within the gastrointestinal tract (7, 8, 14), make this a

particularly intriguing gene to investigate.

Despite all of the unknown aspects of probiotics, there is substantial clinical and
experimental evidence of their beneficial effects. L. reuteri appears to have great
promise as probiotic species, exhibiting many of the in vitro characteristics
classically considered to be “necessary” for a strain to cause beneficial effects,
such as being of host origin, surviving stresses present in the gastrointestinal
tract, and producing antimicrobial compounds. Importantly there are also
multiple L. reuteri strains exhibiting beneficial effects in clinical trials, including
RC-14’s effects on urogenital health (1, 2, 6) and ATCC 55730’s effects on
general health and diarrhea in children (10-13). A better understanding of how L.
reuteri responds to situations within the host and possibly modifies the responses

of the host may lead to further applications for this species.

156

 

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Shornikova, A. V., I. A. Casas, E. lsolauri, H. Mykkanen, and T.
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Wilson, C. L., A. J. Ouellette, D. P. Satchell, T. Ayabe, Y. S. Lopez- _ , .
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158

 

APPENDIX A

MICROARRAY ANALYSIS OF LACTOBACILLUS REUTERI ATCC 55730

DURING MID-LOG AND EARLY STATIONARY PHASES OF GROWTH

PURPOSE

The purpose of this investigation was to compare the gene expression profiles of
a commonly used probiotic strain, L. reuteri ATCC 55730, during mid-log and
early stationary phases of growth. Collaborators at Baylor College of Medicine
identified that certain strains of L. reuteri produce compounds that are able to
suppress TNF production by activated macrophages (as described in Chapter 4).
This activity is found in cell-free conditioned medium isolated from cells at the
stationary phase of growth. The microarray studies described herein were
performed in an attempt to identify potential targets to investigate as the possible
immunomodulatory compounds. L. reuteri ATCC 55730 is not one of the strains
that exhibits the TNF-suppressive activity, yet it was the only sequenced strain at

the time of the experiments.

MATERIALS AND METHODS

Bacterial strains and growth conditions. L. reuteri ATCC 55370 was grown in

MRS broth (BD Difco) at 37°C under microaerobic conditions (2% 02, and 5%

159

 

w: “Mi-"j

 

002, balanced with N2) with slow shaking, or grown on MRS agar plates
incubated anaerobically using the GasPack EZ Anaerobe Container system (BD

Difco) at 37°C unless otherwise specified.

RNA isolation. L. reuten' ATCC 55730 from an 18-hour old culture was
inoculated into 40 ml of MRS broth to a starting 0.0.500 approximately equal to
0.03. Cultures were grown to late stationary phase. For each of the five
biological replicate experiments, 5 ml samples were collected at mid-log phase
(O.D.600 approximately equal to 0.3) and early stationary phase (0.0.600
approximately equal to 2.4) and immediately mixed with an equal portion of ice-
cold methanol to halt transcription. Cells were pelleted by centrifugation at 4000
rpm for 10 minutes and washed with STE buffer (6.7% sucrose, 50 mM Tris-Cl,
pH 8.0, 1 mM EDTA). The washed cells were resuspended in 0.25 UhltS/lll
mutanolysin (Sigma) dissolved in STE buffer and incubated at 37°C for 20
minutes. RNA was then isolated from lysed cells using the Qiagen RNeasy Kit
with optional on column DNase treatment according to manufacturer’s

instructions.

Microarray experiments. Microarray experiments were performed as previously
described (5). In short, 60-mer oligonucleotides for 1864 open reading frames
were synthesized for the L. reuteri ATCC 55730 genome (1 ); 15 open reading
frames from L. reuteri DSM20016 encoding extracellular proteins were also

incuded. Various E. coli genes with no known similarity to the L. reuteri genome

160

 

were included as controls. The synthesized oligonucleotideswere spotted onto
Corning UltraGAPS-ll slides; with each gene represented once on the
microarray, while the control spots were spotted eight times, once in each
subgrid. All work, from the design of the oligonucleotides to the array
construction were performed at the Research Technology Support Facility at
Michigan State University, East Lansing, MI, USA. RNA isolation, labeling, and
hybridization were performed as previously described (3, 4). (Information
regarding the microarray platform can be found at NCBls Gene Expression
Omnibus (GEO, h_ttp://www.ncbi.nlm.nih.gov/geo/) under GEO platform number

GPL6366).

Five biological replicates, along with technical replication through dye-swapping,
were performed for the microarray experiments. In other words, each RNA
sample was subjected to two hybridizations and values used for subsequent
analysis were averaged. Microarray data was analyzed using iterative outlier

analysis with three iterations as previously described (2, 3).

RESULTS

Microarray experiments were performed to investigate the differences in the gene
expression profiles of L. reuteri ATCC 55730 cultures at mid-log and early
stationary phases of growth. The points of samples collection are shown in

Figure A.1, which shows a representative growth curve for the strain. Overall, 85

161

 

genes were found to be significantly over-expressed as the cultures entered
stationary phase, and 41 genes were found to be significantly under-expressed
based on the iterative outlier analysis. A list of genes with significantly different

expression patterns can be found in Tables A1 to A2.

 

 

 

10 -
1 _
O
.8
Q
o
0.1 ~
0.01 T l l. l
0 100 200 300 400
time (min)

 

 

 

Figure A.1. A representative growth curve of L. reuten' ATCC 55730. Samples
for microarray analysis (mid-log and early stationary phase) were taken at the
time point represented by the open circles.

162

 

 

Table A.1. Genes significantly over-expressed by L. reuteri ATCC 55730 during
early stationary phase.

 

 

 

 

 

 

 

 

 

163

Accession
Gene a Fold-
name no. Functional classification change_
Energy production and conversion

lr0904 NS Malate dehydrogenase (EC 1.1.1.37) 5.1

Ir1475 NS Malolactic enzyme 2.6

Ir1744 NS L-2-hydroxyisocaproate dehydrogenase 4.9
bifunctional protein: alcohol
dehydrogenase; acetaldehyde

lr1777 EF421920 dehydrogenase 3.5

lr1793 EF421923 1,3-propanediol dehydrogenase 3.2

lr1866 D0233710 PduW putative acetate kinase 7.6
COG1454 EutG Alcohol

lr1867 D0233711 dehydrogenase class IV 11.6
PduP putative NAD-dependent

Ir1868 D0233712 aldehyde dehydrogenase 10.3
PduA propanediol utilization protein:

Ir1882 D0233726 putative microcompartment protein 4.9

Ir1919 NS Malate dehydrogenase (EC 1.1.1.37) 2.9

Amino acid transport and metabolism

lr0054 NS Homoserine kinase (EC 2.7.1.39) 2.9

Ir0124 NS Amino acid permease 4.6

lr0125 EF537904 aminotransferase 6.1

lr0213 NS Aminotransferase 6.3
Cystathionine beta-Iyase/cystathionine

lr0324 EF421 866 gamma-synthase 6.0

lr0529 NS Arginine/ornithine antiporter 5.6

lr0530 NS Arginine/ornithine antiporter 4.0

Ir0560 NS Cysteine synthase (EC 4.2.99.8) 8.5
Transcriptional regulator, GntR family /
TYROSINE AMINOTRANSFERASE

lr0621 NS (EC 2.6.1.5) 2.7
4-aminobutyrate aminotransferase (EC

Ir0650 NS 2.6.1.19) 3.8

Ir0701 NS Aspartokinase (EC 2.7.2.4) 4.2

lr1019 DQ857773 ArcC, Carbamate kinase 4.7
COGOO78 ArgF Ornithine

Ir1020 D0233707 carbamoyltransferase partial CDS 7.2

lr1113 EF534269 Threonine synthase 2.7

 

 

 

 

Table A.1 (cont’d)

Aspartate aminotransferase (EC

 

 

 

 

 

 

 

 

 

lr1162 NS 2.6.1.1) 5.1
Ir1258 EF421900 Xaa-Pro aminopeptidase, PepP 3.8
Ir1517 D0233695 COGZZ35 ArcA Arginine deiminase 6.6
COG0624 ArgE Acetylornithine
deacetylase/Succinyl-diaminopimelate
desuccinylase and related deacylases
Ir1731 DQ233708 partial CDS 7.9
Ir1784 D0857803 Peptidase U34 3.1
PduU putative ethanolamine utilisation
Ir1865 DQ233709 protein 9.8
Ir1881 DQ233725 PduB propanediol utilization protein 8.0
Carbamoylphosphate synthase small
Ir2088 D0857861 subunit 2.6
Nucleotide transport and metabolism
Dihydroorotase and related cyclic
Ir0302 D0857868 amidohydrolases 4.6
Ir0521 DQ857762 Dihydroorotate dehydrogenase 3.0
Carbohydrate transport and metabolism
lr1525 EF534272 Arabinose efflux permease 3.1
CobC, AIpha-ribazole-5‘-phosphate
Ir1985 DQ857854 phosphatase 3.2
Coenzyme transport and metabolism
2-dehydropantoate 2-reductase (EC
lr0969 NS 1.1.1.169) 2.8
Lipid transport and metabolism
lr0149 NS Putative lipase/esterase 2.7
(acyl-carrier-protein) S-
lr1009 EF421883 malonyltransferase, FabD 24.4
3-oxoacyI-[acyl-carrier-proteinj
Ir1011 EF421885 synthase Ill, FabH 5.9
3-hydroxymyristoyl/3-hydroxydecanoyl-
Ir1013 EF421887 (acyl carrier protein) dehydratase, FabA 23.7
EnoyI-[acyl-carrier protein] reductase
Ir1279 EF421902 (NADH), Fabl 19.1
AcetyI-CoA carboxylase alpha subunit,
lr1280 EF421903 AccA 16.3
AcetyI-CoA carboxylase beta subunit,
lr1281 EF421904 Ach 14.3
Ir1282 EF421905 Biotin carboxylase, AccC 20.7
3-hydroxymyristoyl/3-hydroxydecanoyl-
Ir1283 EF421906 (acyl carrier protein), FabA 16.1

164

 

lf’fflLfln .- u— .- j

 

 

 

Table A.1 (cont’d)

 

 

 

 

 

 

 

 

 

 

 

 

 

lr1284 EF421907 Biotin carboxyl carrier protein, AccB 18.1
3-oxoacyI-(acyl-carrier—protein)
|r1286 EF421908 synthase, FabB 21.4
3-oxoacyI-(acyl-carrier—protein)
Ir1287 EF421909 reductase, FabG 19.5
Transcription
lr1012 EF421886 Transcriptional regulator, MarR family 20.7
Ir1310 EF421911 hypothetical protein , 4.2
COG1438 ArgR Arginine repressor
lr1518 DQ233704 partial CDS 5.2
lr1573 NS Leucine-responsive regulatory protein 3.2
Replication, recombination and repair
ATPase related to the helicase subunit
lr0044 EF421958 of the Hollidayjunction resolvase 4.7
Posttranslational modification, protein turnover, chaperones
lr0107 NS Thioredoxin 6.1
Inorganic ion transport and metabolism
COG1464 NIpA ABC-type metal ion
transport system periplasmic
lr0629 DQO74834 component/surface antigen 3.6
COG1464 NlpA ABC-type metal ion
transport system periplasmic
Ir1151 DQ074882 component/surface antigen 3.7
lr2077 NS Copper-exporting ATPase (EC 3.6.3.4) 2.6
Secondary metabolites biosynthesis, transport and catabolism
2-oxo—hepta-3-ene-1,7-dioic acid
lr1745 NS hydratase 3.3
lr1873 DQ233717 PduL propanediol utilisation protein 9.6
PduE propanediol dehydratase small
Ir1878 DQ233722 subunit 4.6
PduC propanediol dehydratase large
lr1880 DQ233724 subunit 8.7
PduN propanediol utilization protein:
Ir1871 DQ233715 putative microcompartment protein 7.2
PduJ propanediol utilization protein:
lr1874 DQ233718 putative microcompartment protein 4.5
PduK propanediol utilization protein:
|r1875 DQ233719 putative microcompartment protein 1 1.3
General function prediction only
|r0079 EF471975 D-lactate dehydrogenase 6.1
lr0272 EF537899 flavodoxin 5.6
Ir0313 EF421952 HAD-superfamily hydrolase 2.6

165

 

 

 

Table A.1 (cont’d)

 

 

 

 

 

Ir11 15 NS NAD-dependent oxidoreductase 3.4

Ir1163 NS Nitrilase (EC 3.5.5.1) 4.0

lr1834 NS Nitrilase (EC 3.5.5.1) 4.8

lr1869 DQ233713 PduObis propanediol utilization protein 9.2

Signal transduction mechanisms

lr0628 DQZ33673 LuxS protein 3.7
COG0834 HisJ ABC-type amino acid
transport/signal transduction systems

Ir0793 DQ074847 periplasmic component/domain 4.2

Ir2123 D0857864 Protein-tyrosine phosphatase 8.7

Unknown function

Ir1010 EF421884 Acyl carrier protein, AcpP 12.4
Uncharacterized conserved protein,

|r1047 EF421890 DegV 3.7

lr1319 DQ074896 COG4716 Myosin-crossreactive antigen 3.7

Ir1515 DQ074905 Unknown extracellular protein 2.9
PduO putative propanediol utilization

lr1870 DQ233714 B12 related protein 3.8

Ir1872 DQ233716 PduM propanediol utilization protein 10.9
PduH putative diol dehydratase

Ir1876 DQ233720 reactivation protein 6.1
PduG putative diol dehydratase

lr1877 DQ233721 reactivation protein 7.2
PduD propanediol dehydratase medium

lr1879 DQ233723 subunit 8.1

Ir2078 NS unknown protein 3.9

 

a GenBank accesion numbers are provided (NS = not submitted)

b Genes were classified based on COG domains found in the protein

sequence through a
search of the JGI Integrated Microbial Genomes database.

166

 

 

 

Table A2. Genes significantly under-expressed by L. reuteri ATCC 55730

during early stationary phase.

 

 

 

 

 

 

 

 

 

 

 

 

 

Accession
Gene a Fold-
name no. Functional classification change
Energy production and conversion
[Citrate [pro-3S]-lyase] ligase (EC
lr0599 DQZ33700 6.2.1 .22) 5.0
NAD-dependent malic enzyme (EC
Ir0600 DQ240820 1.1.1.38) 5.6
COG3051 CitF Citrate lyase alpha
Ir1418 DQ233691 subunit 4.3
Amino acid transport and metabolism
spermidine/putrescine ABC
Ir0137 EF537897 transporter, permease protein 5.3
Glutamine transport ATP-binding
lr1197 NS protein gan 5.1
COGO834 HisJ ABC-type amino acid
transport/signal transduction systems
periplasmic component/domain
COG0765 HisM ABC-type amino acid
transport system permease
lr1 198 DQ074887 component 7.0
Ir1508 NS Gluconate permease 2.9
Histidine transport ATP-binding
Ir1664 NS protein hisP 3.2
amino acid permease-associated
N886:1 NS region 3.0
Nucleotide transport and metabolism
lr0092 EF421961 Deoxyribose-phosphate aldolase 3.2
Ir0115 DQ219946 COG1428 Deoxynucleoside kinase 4.1
Pyrimidine-nucleoside phosphorylase
Ir0674 NS (EC 2.4.2.2) 3.3
Carbohydrate transport and metabolism
lr0101 EF421948 Phosphopentomutase (EC 5.4.2.7) 2.9
lr0160 EF547651 Sugar kinase, ribokinase family 13.7
lr0590 NS Deoxyribose transporter 3.9
lr0675 00857878 Phosphopentomutase 2.9
COG2301 CitE Citrate lyase beta
lr1419 DQ233692 subunit 5.8
NS359:2 NS Ribose transport protein 12.6

 

 

167

 

 

Table A2 (cont'd)

 

Coenzyme transport and metabolism

 

COG1767 CitG Triphosphoribosyl-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ir1417 DQZ33690 dephosgho-COA synthetase 5.4
Lipid transport and metabolism
N8207z1 NS Transcription regulator 5.8
Translation, ribosomal structure and biogenesis
Ir0050 NS LSU ribosomal protein L14P 2.9
C060594 RnpA RNase P protein
lr0251 DQ219950 component 3.2
tRNA (Guanine-N(1)-)-
lr1551 NS methyltransferase (EC 2.1.1.31) 3.5
Transcription
lr0647 NS Catabolite control protein A 3.2
Replication, recombination and repair
lr1237 NS DNA gyrase subunit A (EC 5.99.1.3) 3.6
lr1238 NS DNA gyrase subunit A (EC 5.99.1.3) 3.4
lr1239 NS DNA gyrase subunit B (EC 5.99.1.3) 3.6
COG1195 RecF Recombinational
lr1240 DQ219962 DNA repair ATPase 4.1
Cell motility
putative type II secretion system
NS376:1 NS protein 7.0
General function prediction only
|r0517 NS Zinc protease (EC 3.4.99.-) 3.2
COG0596 MhpC Predicted
hydrolases or acyltransferases
Ir1416 DQZ33689 (alpha/beta hydrolase superfamily) 3.2
|r1666 NS putative zinc-dependent peptidase 4.2
N8337z1 NS Conserved hypothetical protein 4.3
Signal transduction mechanisms
Stress response membrane GTPase
Ir0279 EF421950 TypA 3.0
COG0834 HisJ ABC-type amino acid
transport/signal transduction systems
Ir1665 DQO74920 periplasmic component/domain 3.5
Intracellular trafficking secretion, and vesicular transport
COG0706 Preprotein translocase
|r0252 DQO74813 subunit YidC 3.0
Unknown function
Ir0646 DQ233675 Hypothetical membrane protein 3.0
lr0982 D0857874 Hypothetical protein 3.4

168

 

 

Table A2 (cont’d)
Ir1434 DQ074902 Unknown extracellular protein 3.1
lr1487 DQ074904 Unknown extracellular protein 4.1
NS104:1 NS unknown protein 3.4

 

a GenBank accesion numbers are provided (NS = not submitted)
b Genes were classified based on COG domains found in the protein
sequence through a

search of the JGI Integrated Microbial Genomes database.

The microarray experiments described were performed to identify potential
immunomodulatory compounds based on gene expression changes as L. reuteri
transitions from mid-log to early stationary phases of growth. Although L. reuteri
ATCC 55730 is not one of the strains capable of producing compounds that
suppress TNF production, it was the only strain for which sequence information
and therefore, microarrays, were available at the time of the study. The
microarray studies revealed two main pathways that were up-regulated as the
bacteria transitioned from mid-log to early stationary phase, propanediol
utilization and fatty acid biosynthesis. The interest in fatty acid biosynthesis led
to the identification of fatty acid differences between the immunomodulatory and

immunoneutral strains as described in Chapter 4.

This data is not included in Chapter 4 because preliminary experiments carried
out by the lab at Baylor College of Medicine using microarrays designed for one
of the immunomodulatory strains, L. reuteri ATCC PTA 6475, did not confirm
similar gene expression changes for this strain. This may be due to genuine

differences in gene expression between the two strains or due to the collection of

169

 

 

samples at different time points (and possibly different stages of growth) between

the two labs.

170

 

REFERENCES

Bath, K., S. Roos, T. Wall, and H. Jonsson. 2005. The cell surface of
Lactobacillus reuteri ATCC 55730 highlighted by identification of 126
extracellular proteins from the genome sequence. FEMS Microbiol. Lett.
253:75-82.

Britton, R. A., P. Eichenberger, J. E. Gonzalez-Pastor, P. Fawcett, R.
Monson, R. Losick, and A. D. Grossman. 2002. Genome-wide analysis
of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis.
J. Bacteriol. 184:4881-90.

Uicker, W. C., L. Schaefer, and R. A. Britton. 2006. The essential
GTPase RbgA (quF) is required for 508 ribosome assembly in Bacillus
subtilis. Mol. Microbiol. 59:528-40.

Wall, T., K. Bath, R. A. Britton, H. Jonsson, J. Versalovic, and S.
Roos. 2007. The early response to acid shock in Lactobacillus reuten'

involves the CIpL chaperone and a putative cell wall-altering esterase.
Appl. Environ. Microbiol. 73:3924-3935.

Whitehead, K., J. Versalovic, S. Roos, and R. A. Britton. 2008.
Genomic and genetic characterization of the bile stress response of
probiotic Lactobacillus reuteri ATCC 55730. Appl. Environ. Microbiol.
74:1812-1819.

171

 

APPENDIX B

IDENTIFICATION OF A PUTATIVE BILE-INDUCIBLE TRANSCRIPTIONAL

ELEMENT FROM LACTOBACILLUS REUTERI

PURPOSE

Bile has been demonstrated to serve as a signal for particular bacteria regarding

 

their location in the gastrointestinal tract. For example, certain pathogens, 3, ._
including Vibrio, Shigella, Salmonella, and Campy/obacter, are known to alter ’
expression of virulence factors such as toxin production, motility, and type III
secretion systems in response to bile (2). Various bile acids have also been
shown to affect the germination of Clostridium difficilespores (3, 4). As there is
known to be overlap in the interaction of pathogenic and commensal bacteria
with the host, It is possible that bile also acts as a signal for probiotic bacteria.
Using the microarray analysis of the bile shock response of L. reuteri ATCC
55730 (5), the purpose of this study was to use a bioinformatic approach to
identify a bile-inducible transcriptional element associated with promoters

induced by bile.

172

 

RESULTS

The upstream regions (300 bp) of all genes over-expressed during the bile shock
response of L. reuteri ATCC 55730 were analyzed for over-represented motifs
using the freely-available internet program MEME (1). As it is unlikely that all of
the genes over-expressed during bile shock will be regulated by the same
element, multiple MEME runs were performed comparing the upstream regions
of smaller subsets of genes. One statistically significant 13 base pair motif was
identified when the upstream regions of 13 genes over-expressed during bile
shock were compared. The upstream regions included in this particular run were
from the following genes: lr0359, lr0540, lr0685, lr0783, lr0922, Ir1067, Ir1139,
Ir1351, lr1468, Ir1684, lr1786, lr1864, and Ir1937. The sequence of the motif at
each of these locations, as well as the consensus sequence is shown in Figure

8.1.

173

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Gene
number Motif sequence
|r0359 CAATTGCGTCATC
Ir1067 CAATTGCTTGATC
|r1786 CAATTGCATAATC
Ir0685 CAATTGGGTTATC
lr1351 CAGTTGCCTTATC
Ir0922 CATTTGCTTCATC
lr1468 GAATTGCATCATC
lr0783 CAATTGGGTGAAC
lr1139 CAATTGCATAATT
Ir1937 CAATTCCTTCATG
lr1684 CCGTCGCGTTATC
|r0540 CAATTGCCAAATG
lr1864 TAAATGCGTTAAC
[ConsensuslclAIAlrlrlclcGTCAITIcI

A T

T A

 

 

 

 

 

 

Figure 8.1. The sequences of the 13 occurrences of the possible bile-inducible
transcriptional element in L. reuteri ATCC 55730 identified by MEME.

In collaboration with C. Titus Brown at Michigan State University, genome-wide
analysis of this putative bile-inducible transcriptional element was performed. Dr.
Brown identified that this particular 13 base pair sequence was found a total of
70 times throughout the L. reuteri ATCC 55730 genome and 54 times throughout
the genome of another probiotic strain, L. reuteri ATCC PTA 6475. If this were a
random sequence element it would be expected to be found only 11 times in a
genome with the size (approximately 2.3 mega-bases) and GC content of L.

reuteri ATCC 55730.

174

 

Further analysis revealed that of the 70 occurrences of this particular 13 base
pair element in L. reuteri ATCC 55730, 66 of them are within the coding regions
of other genes. It was also demonstrated that none of the additional 57 elements
(beyond the original 13 identified) are located upstream of genes that are
significantly over-expressed or under-expressed during the bile shock response

of this strain.

FUTURE DIRECTIONS

Current work in the lab is focused on developing a reporter gene system for use
in L. reuteri. This system will ultimately allow us to identify whether this putative
13 base pair transcriptional element is a bile-inducible promoter. A bile-inducible
promoter could have great potential for application in the probiotic industry, as it

would allow for targeted expression of a gene product in the host small intestine.

175

 

REFERENCES

Bailey, T. L., and C. Elkan. 1994. Presented at the Proceedings of the
Second International Conference on Intelligent Systems for Molecular
Biology, Menlo Park, California.

Gunn, J. S. 2000. Mechanisms of bacterial resistance and response to
bile. Microbes and Infection 2:907-913.

Sorg, J. A., and A. L. Sonenshein. 2008. Bile salts and glycine as
cogerminants for Clostridium difficile spores. J. Bacteriol. 190:2505-2512.

Sorg, J. A., and A. L. Sonenshein. 2009. Chenodeoxycholate is an
inhibitor of Clostridium difficile spore germination. J. Bacteriol. 191:1115-
1 1 17.

Whitehead, K., J. Versalovic, S. Roos, and R. A. Britton. 2008.
Genomic and genetic characterization of the bile stress response of
probiotic Lactobacillus reuteri ATCC 55730. Appl. Environ. Microbiol.
74:1812-1819.

176

 

APPENDIX C

INVESTIGATION OF A PUTATIVE MATRIX METALLOPROTEASE FROM

LAC TOBA CILLUS REUTERI
PURPOSE

The purpose of this study was to investigate the role of a putative
metalloprotease identified in Lactobacillus reuteri. This gene, lr1291, was
identified as being significantly over-expressed during the bile shock response of
L. reuteri ATCC 55730. Mutational analysis did not reveal a role for this gene in
survival or growth in the presence of bile (14). Sequence analysis revealed that
' the protein has significant similarity to eukaryotic matrix metalloproteases
(MMPs), a class of proteins known to be involved in a wide range of important
functions such as recruitment of immune cells, cytokine and chemokine
activation, and degradation of the extracellular matrix. As very little is currently
understood regarding how probiotic bacteria interact with the host to cause their
beneficial effects, the presence of a gene with significant similarity to eukaryotic

proteins known to play important roles in the gastrointestinal tract is of interest.
Note: Although the gene was originally identified in L. reuteri ATCC 55730

(Ir1291), the homolog found in L. reuteri ATCC PTA 6475 (NT01LR0595) was

used for all bioinformatic and over-expression studies discussed herein. There is

177

 

 

96% similarity between Lr1291 and NT01LR0595 at the amino acid level, and
93% similarity at the nucleic acid level; both proteins are referred to as Lr1291 for

simplicity.
BIOINFORMATICS

A BLAST of the protein sequence of Lr1291 against the non-redundant protein
sequences in GenBank revealed that the closest matches were peptidases in the
M10A or M12B family (including matrixins and adamalysins), putative
metalloproteinases, or zinc-dependent proteases in either Lactobacillus spp. or
Streptococcus spp (1 ). Lactic acid bacteria with similar proteins included
primarily host-associated strains such as lactobacilli commonly used as
probiotics, including L. reuteri, L. acidophilus, L. gasseri, L. johnsonii, and L.
plantarum, as well as pathogenic streptococci including Streptococcus
pneumoniae and S. mutans. The significant hits also included several human
matrix metalloproteases (MMPs) that aligned with the second half of Lr1291. A
search of the conserved domain database (5) revealed that the last 130 amino
acids of Lr1291 contains cd04268 (ZnMc_MMP_Iike subfamily). This group
includes matrix metalloproteases, serralysins, and astacin-Iike family of

proteases.

To further investigate the sequence of Lr1291, the protein sequence was

compared to the MEROPS database (10). This search revealed that Lr1291 fits

178

 

into the M10 family, although it was classified along with other unassigned
peptidases. The active site for the M10 family is HExxHxxgxxH, including the
histidine residues that act as zinc ligands and the catalytic glutamate. This family
shares the same active site as several other families of metalloproteases (M12,
M43, and M57). The M10 family peptidases are classified as “metzincins”
because of the conserved methionine residue found C-terminal to the active site.
The M10 family peptidases often are also found to function outside of the cell. In

line with this, Lr1291 is predicted to be secreted, due to the presence of an N-

 

terminal signal peptide without any discernable cell envelope anchor (2). Figure
0.1 shows the protein sequence of Lr1291with several characteristics of interest

marked.

>NT01 LR0595

VNEINFIFRLLKRIFIlGLLVGGGWLYFNDARVQA II
TANQTAWNVRDRIAKLIGRDDTNSNDNSNLHLNDANNGSSKNEQGPTTEQQT
STQTSIPSTGRWATNQATVYVNTNNAQLDAATNTAIQNWNQTGAFTFKPVNNQ
SKADIVVTTMNRSDSNAAGLTKTSSNSLTRRFMHATVYLNTYYLTDPSYGYSQE
RIVNTAE HELGHAIGLDH TNAVSV M QPAGSFYTIQPDDVQAVQKLYANNK

Figure C.1. The protein sequence of L. reuteri ATCC PTA 6475 NT01LR0595
(Lr1291 homolog). The putative cleavage site is marked with two forward
slashes (II); this site was predicted by SignalP 3.0 (3, 8). The putative active site
is underlined, with the conserved residues in bold. The conserved methionine
residue (“met-turn”) is also in bold.

These similarities and sequence comparisons suggest that Lr1291 is part of a

novel series of bacterial zinc-dependent metalloproteases of unknown function.

Although there are other known bacterial zinc-dependent metalloproteases, none

179

of these proteins of known function were found to have significant sequence

similarity.
EXPERIMENTS AND RESULTS

This section will briefly outline some of the experimental attempts to determine

the function of Lr1291.

Expression vectors and protein forms. Various expression vectors were used
in attempts to over-express Lr1291. Original cloning attempts were made into
traditional pET vectors, including pET-21b (C-terminal his-tag expression vector)
and pET-19b (N-terminal his-tag expression vector). Due to the difficulty in
cloning (owing to low transformation efficiencies and high background levels, a
pET-21 b construct was never constructed; one pET-19b construct was
constructed after multiple attempts), the Gateway system (Invitrogen) was
employed. Several constructs were made successfully using this system, albeit
at much lower efficiencies than suggested by the Invitrogen product manual. The
vectors successfully used for constructs included: pET-DEST42 (C-terminal his-
tag expression vector), pDEST-17 (N-terminal his-tag expression vector), and
pDEST-15 (N-terminal GST-tag expression vector). Various forms of the gene
were also used: the complete Lr1291 sequence, the Lr1291 sequence beginning

with the putative cleavage site, and the complete Lr1291 sequence with the

180

 

 

 

active site mutated from HExxHxxGxxH to AAxxHxxGxxH. All constructs were

confirmed by sequencing.

Induction strains and conditions. Multiple strains were tested for induction
with the various expression vectors. These included: E. coli BL21, E. coli Tuner,
and E. coli Rosetta (DE3) pLysS cells (Novagen). Induction for these strains with
the various vectors was attempted at 16° C, 25° C, 30° C, and 37° C in the
presence and absence of 100 uM zinc acetate. Cell Iysates from all cultures
were prepared by sonication and loaded onto SDS-PAGE gels. No difference
was detected between proteins from uninduced and induced cultures under any
conditions tested. Lysis of the induced cultures was observed on multiple

instances, suggesting a toxic effect of the protein.

Codon usage. Lr1291 contains multiple codons that are rarely used in E. coli
(including four occurrences of AUA encoding isoleucine and six instances of
GGA encoding glycine). In order to determine if codon usage played a role in the
lack of high expression levels in E. coli BL21, Rosetta cells were employed for
expression. E. coli Rosetta (DE3) pLysS cells from Novagen contain the pRARE
plasmid, which supplies the tRNAs that recognize the following rare codons:
AUA, AGG, AGA, CUA, CCC, and GGA (9). Use of Rosetta cells did not result in

detectable expression levels of Lr1291.

181

.. 7.7
- .
I
,0

 

 

 

Other systems for expression. When multiple attempts were made to express
the protein in E. coli using various vectors, expression strains, and induction
conditions, other systems were employed. These included an in vitro translation
system (Qiagen EastPress Protein Synthesis Kit, catalog number 32501 ), over-
expression in Bacillus subtilis (using the integrative pDR111 plasmid), and over-
expression in Kluyveromyces lactis (New England Biolabs yeast expression kit,
catalog number E10008). None of these systems resulted in detectable over-
expression of Lr1291. When a construct containing Lr1291 was included in the
in vitro translation reaction with the control vector, over-expression of the control
protein (elongation factor EF-Ts) was completely shut down. This result

suggests that production of Lr1291 may interfere with translation.

Isolation from conditioned medium. After attempts at over-expression failed,
experiments were carried out to try to isolate the protein from conditioned
medium of L. reuten' ATCC 55730 and L. reuteri ATCC PTA 6475. For these
experiments, both wild-type strains and mutants for each strain containing a
disruption in Lr1291 were used. Cell-free conditioned medium from cells grown in
deMan, Rogosa, Sharpe (MRS) broth (Difco) were used. Samples were
collected from mid-log phase cultures, before and 15 minutes after exposure to
bile, and from overnight cultures. The cell-free medium was concentrated under
nitrogen, as some component of MRS broth clogged all types of centrifugation

concentration devices tried. Despite using the same conditions where Lr1291

182

A“ 1.11 I,

 

was shown to be over-expressed (15 minutes after bile exposure, Chapter 2) no

consistent protease activity was identified in these samples.

Because of the inconsistencies observed with concentrated conditioned medium
from L. reuteri cultures, attempts were made to precipitate proteins from the
cultures using 20% trichloroacetic acid. The samples were loaded onto SDS-
PAGE gels and onto casein and gelatin zymograms. No difference in the
secreted proteins was detected between the wild-type L. reuteri and the Lr1291
mutant strains. lmportantly, no protease activity was detected in any of the
samples tested. The protocol for precipitation of proteins from L. reuteri

conditioned medium is included at the end of this Appendix.

Detection of activity. In all cases (protein expression and isolation from
conditioned medium), samples were applied to either casein or gelatin
zymograms to determine if any protease activity was detected. In several cases,
protease activity was observed. Further investigation revealed that this protease
activity was present in any E. coli lysate and was not specific to the production of

the protein of interest.
Note: Although some companies sell pre-made zymogram gels, the use of these

products is not recommended. The results obtained with these products were

highly variable. Due to the difficulty and inconsistencies in various zymogram

183

 

 

 

protocols, the protocol listed at the end of this Appendix obtained from Dr. Jenifer

Fenton at Michigan State University is recommended for use.

DISCUSSION

The role of Lr1291, a protease of unknown function with homologs in various
strains of host-associated lactic acid bacteria, should be investigated. The
protein sequence shows intriguing similarity to eukaryotic matrix
metalloproteases, a class of proteins known to play wide-ranging roles in normal
development, inflammation, and the immune response. The MMPs have been
implicated in important roles in the gastrointestinal tract, specifically in the
intestinal immune response, the activation of defensins (antimicrobial peptides
produced in the small intestine) and the development of inflammatory bowel
disease (6, 7, 15). It is possible that proteases like Lr1291 may play an important

role in the interaction of bacteria with the host.

It is difficult to determine why there was a lack of experimental success for this
project. The problems with the protein expression systems (low success rate in
cloning, lysis of induced cultures, etc) suggest a level of protein toxicity, although
the lack of success in over-expression of the mutant protein (active site changed
from HExxHxxGxxH to AAxxHxxGxxH to inhibit activity) suggests that factors
other than the protein activity are involved. A similar protein also found in L.

reuten', Lr1017, did not cause lysis of the expression strains and was shown to

184

A' .'A‘ ’ Kind"?
r.
I

 

 

be over-expressed using a pDEST-17 construct in the E. coli BL21 expression
strain, suggesting that the problem is specific to Lr1291 and not a simple

technical issue.

The lack of activity observed with cell-free culture supernatant and precipitated
secreted proteins from L. reuteri ATCC 55730 and ATCC PTA 6475 could be due
to a low level of protein production under the growth conditions used or the use
of an inappropriate substrate for this particular protease. It is also possible that
the protein in question, Lr1291, is not actually secreted or may be secreted in an

inactive form.

Although casein and gelatin zymography are commonly used to detect general
protease activity, it is possible that this particular protease has a more specific
substrate. Several eukaryotic MMPs have either shown little or no activity
against gelatin in vitro, thus the use of other substrates such as elastin, collagen,
and various synthetic peptides should be considered for future use (4, 11, 12).
Detection of activity from matrilysin (MMP-7) has been particularly difficult, and
sensitive assays using synthetic fiuorogenic peptides as substrates have been
employed (13). A screen of several of these synthetic peptides may be a more

sensitive and possibly useful way of identifying protease activity from Lr1291.

Other difficulties have been encountered in attempts to determine the role of

various eukaryotic proteases. For example, MMP-23 did not demonstrate any

185

,. " 1; n. )

 

 

protease activity against multiple substrates in vitro, suggesting either unique
substrate specificity or a mis-folding of the protein during purification. Weak
protease activity was finally achieved with a chimeric protein, combining the
propeptide domain of MMP-19 (a metalloprotease known to autoactivate) with
the catalytic domain of MMP-23, suggesting that activation of the protein
(cleavage of the prodomain) may have been an issue in vitro (12). Despite the
multiple attempts in this study at detecting activity from cultures of E. coli induced
to express the protein and from conditioned medium of L. reuteri, mis-folding or a
lack of proper activation of Lr1291 are possibilities that should also be

investigated.

Current work in the lab by another researcher suggests that some success in
over-expressing the cleaved form of Lr1291 may be found using a Lactococcus
lactis expression system. If this finding is confirmed, careful selection of a wide-
range of substrates and sensitive assays should be employed for detection of
protease activity. The value of the mutant strains of L. reuteri containing
disrupted Lr1291 genes should also not be ignored. lf attempts to purify the
protein are not successful, these strains could be employed in various assays,
such as activation of defensins or production of cytokines and chemokines by

eukaryotic cells, in attempts to determine the role of this protease.

186

 

 

PROTOCOLS OF INTEREST

Protein precipitation from L. reuteri conditioned medium.

1. Grow L. reuten' in LDM-lll broth containing Tween 80 and glucose (MRS broth

contains too many proteins; the background levels of precipitated proteins will

 

interfere with results). At desired time, collect cells by centrifugation at room I}

temperature for 10 minutes at 4000 rpm. Filter supernatant through a 0.22 (M

 

filter into a 50 mL conical tube. I ’

2. Add ice-cold 100% trichloroacetic acid (TCA) to a final concentration of 20%.

Incubate on ice at 4° C (cold-room) for 48 hours.

3. Transfer mixture to Sorvall tubes and spin at 12,000 x g for 30 minutes at 4° C.

Remove supernatant.

4. Resuspend pelled in 2 mL of ice-cold acetone (if starting with original culture
volume of approximately 20 mL). Incubate on ice for 30 minutes. Transfer

mixture to two 1.5 mL eppendorf tubes.
5. Spin at maximum speed in a microcentrifuge kept at 4° C for 30 minutes.

Remove supernatant and air-dry pellets for 2 to 3 minutes. Resuspend pellet in

desired sample buffer and proceed. Note: For zymograms, do not resuspend

187

 

pellets in the LDS sample buffer provided for use with the pre-cast Invitrogen
protein gels. Although this buffer works well for visualization of the proteins, it

will destroy any protease activity present in the samples.

188

u .u ‘Jfljl' a
- I

 

 

Zymograms (SDS-PAGE Substrate Gel Method)

Stock Solutions
- 30% Acrylamidelbis-Acrylamide (29:1) solution (purchase from Bio-rad,
catalog #161-0156). Store in refrigerator.
- 2.0 M Tris, pH 8.8. Store in refrigerator
- 0.5 M Tris, pH 6.8. Store in refrigerator.
- 0.625 M Tris, pH 6.8. Store in refrigerator.
-10% 803 stock. Store at room temperature.
-10% ammonium persulfate. Weigh 0.1 g ammonium persulfate in a
microcentrifuge tube, add 1 mL of ddH20 the day of use, throw away when done.
- Substrate - Gelatin: 2.0 mgImL Swine skin gelatin (Sigma, #G2500). Weigh
100 mg gelatin in a 50 mL conical tube, add 50 mL of ddH20, warm slightly to
dissolve gelatin completely in water. Store in refrigerator.
- Substrate - Casein: 2.0mglmL casein in 5mM Tris (Sigma, #07078).
Dissolve 100mg of casein in 20 mL of 10mM NaOH and heat to get in solution
while stirring. Neutralize by slowly dripping in 20mL of 10mM HCI while stirring
on ice (sep funnel). Add 500 mM Tris, pH 7.5 (.5mL). Add 9.5 mL ddHZO. Store
in refrigerator for no more than a month.

*may not have to add the entire 20 mL of 10 mM HCI. Add slowly,
stopping as soon as the solution becomes slightly cloudy (usually somewhere

between 14 to 20 mL in my experience). Can make up any needed volume by

189

 

 

 

the addition of more ddH20. The addition of excess HCI will cause the casein to
crash out of solution.

- 4X Sample buffer (40% glycerol, 4% SDS, and 0.25 M Tris; pH 6.8). For 10
mL total volume, add in this order to a 15 mL conical tube: 0.4 9 SDS, 4 mL of
0.625 M Tris stock, 2 mL ddH20. Vortex. Add 4 mL glycerol and a small amount
of powdered bromophenol blue. Vortex.

- Running buffer (total volume of 4 mL). Add 1 L ddH20 and a stir bar to a 2 L
graduated cylinder, weigh out 57.6 g glycine, 12.1 g Tris, and 4.0 9 SDS, add the
three chemicals to the 2 L graduated cylinder, fill the graduated cylinder to the 2
L mark and stir until clear. Pour into a 4 L amber bottle. Add an addition 2 L of
ddH20 to the amber bottle, cap, and shake.

- Equilibration buffer - 2.5% Triton-X100 (total volume of 4 mL). Add 1.9 L
ddH20 and a stir bar to a 2 L graduated cylinder place on a warm plate (medium
setting) and stir, add 100 mL of Triton-X100 very slowly. Stir until clear. Pour into
a 4 L amber bottle. Add an additional 2 L of ddH20 to the amber bottle, cap, and
shake.

- Incubation Buffer. 50 mM Tris HCI pH 7.5, 200 mM NaCl, 10 mM CaCI2, 10
uM ZnCI2, 0.02% Brij 35 (only good for one week once the calcium and zinc are
added)

- 4X Stain. Add 1 g Coomassie blue R250 to 1L methanol, stir and gravity filter.

-1X Stain. Add to a 500 mL glass bottle, 100 mL 4X stain, 40 mL glacial acetic

acid, and 260 mL ddH20, shake.

190

 

 

 

Procedure (Mini gels = 7.5 mL)

Day 1
1. Substrate gel

a. Mix the following in a conical vial for one gel:

2.0 mL 30% acrylamide stock

150 uL 10% SDS

1.4 mL 2.0 M Tris, pH 8.8

2.13 mL ddH20

1.86 mL gelatin stock or casein stock

 

b. Add together simultaneously to mixture above:
83.6 ul ammonium persulfate

10.7 m TEMED

0. Mix pour immediately by slowly adding liquid gel to chamber with a
transfer pipette. Slowly overlay gel with approximately 320 uL ddH20 (160

“L twice).

d. Let stand at room temperature for 1 hour (remove water overlay before

pouring stacker).

2. 4 % Stacking gel

a. Mix the following in a conical vial for two stackers:

191

 

0.65 mL acrylamide
1.25 mL 0.5 M Tris, pH 6.8
50 mL 10% SDS

3.05 mL ddH20

D. Add together simultaneously to mixture above:

 

 

25 mL ammonium persulfate

6 mL TEMED

 

c. Center comb in chamber and add liquid stacking gel with a transfer
pipette, be careful not to create air bubbles. Let sit at room temperature

for 30 minutes.

3. Loading the gel

a. Enzyme samples are mixed with 4X sample buffer (32 mL sample: 8 mL

 

sample buffer); load 40 (LL into well.

b. For molecular weight ladders follow the protocol for each.

c. Load samples with pipette tips while gels are completely submersed in

running buffer, use a new tip for each sample. Check that the inner area is

completely filled with running buffer and that the outer area is at least

covered by buffer to the top black knob with running buffer. You can use a

192

transfer pipette to fill the inner area of the apparatus. This allows good

current flow.

4. Running the gel

Be sure that the red electrode is connected to the red outlet and the black
to the black. Run the gel at 65 V until the samples are thru the stacking gel
(approximately 30 minutes); then at 120 V (or 100 V if separation looks
odd) until the blue line is below the gray seal at the bottom of the glass
plates. Be sure that the protein does not run off the plates. While running
the gel watch that the samples run at the same speed; watch for “smile” or
“frown” sample lines. If they start to form, either increase or decrease the

voltage to eliminate the “smile” or “frown”.

5. Preparing the gel for staining
a. Once the samples are done, remove the gel apparatus from the buffer
chamber set-up. Remove the gel from between the glass plates. Carefully
remove the plates from their position on the gel apparatus. Gently lift one
spacer (to release the vacuum that forms while running the gel between
the plates) to remove the top plate (shorter plate). The gel will remain

attached to the top (shorter) plate.

b. Remove the stacker from the gel by pressing a piece of paper towel

along the stacker portion of the stacker-gel junction. Next, gently pull up

193

 

on the paper towel to remove the stacker from the gel. Cut the upper right
hand corner so that the orientation of the gel is known. By cutting the right
top corner you are cutting the area where the marker is loaded or lane lot
the gel. If more than one gel is run, out two (same side) or three corners

so that the gels can be distinguished from each other.

0. Place each gel in a container, submerse each gel in equilibration buffer
and cover container. Shake for 30 minutes in rocking water bath. Pour off
old buffer in sink (be sure to retain gels in the container). Add new
equilibration buffer to submerse each gel and cover container. Shake for
30 minutes in rocking water bath. Pour off buffer in sink (be sure to retain
gels in the container). Add incubation buffer to submerse gel, cover

container and incubate at 37C overnight (or for up to 48 hours).

Day 2
6. Staining the gel
Stain: gel for 1 hour with 1X stain: (completely submerse gel) at room

temperature. Rock on rocker in covered container.

7. Destain

Destain the gel in ddH20.

194

 

8. Storage of the gel

Store gel in 10% glycerol or ddH20.

195

 

10.

11.

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