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thesis entitled
THE EFFECT OF SURFACTANTS UPON THE ACTIVITY

AND DISTRIBUTION OF GLUCOSYLTRANSFERASE

IN STREPTOCOCCUS MUTANS 6 l
md‘b'y— 7 5

pr:

Catherine Wernette

has been accepted towards fulfillment
of the requirements for

MASTER OF SCIENCE degreein Microbiology and
Public Health

 

 

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Major professor

Date JQVfo/wg... 7f/

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THE EFFECT OF SURFACTANTS UPON THE ACTIVITY
AND DISTRIBUTION OF GLUCOSYLTRANSFERASE

IN STREPTOCOCCUS MUTANS 6715

 

By

Catherine Wernette

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and
Public Health

1981

ABSTRACT

THE EFFECT OF SURFACTANTS UPON THE ACTIVITY
AND DISTRIBUTION OF-GLUCOSYUTRANSFERASE
IN STREPTOCOCCUS MUTANS 6715

By
Catherine wernette

 

A modified Somogyi—Nelson procedure was used to determine the
amount of reducing sugar produced during the reaction of Streptococcus
mgt§n§_6715 glucosyltransferase (GTF) with sucrose in the presence of
the surfactants glycerol monolaurin (GML) and sodium lauryl sulfate (SLS).
GML and SLS both reduced extracellular culture filtrate GTF enzyme

activity.’ Secondly, Streptococcus mutans 6715 cultures were grown in

 

medium.containing GML, Tween 80, or SLS. 'Whole culture GTF activity was
decreased when the organism was grown in the presence of GML. TWeen 80
caused a large increase in whole culture GTF activity, whereas SLS

resulted in no change in the GTF activity as compared to the control.

TABLE OF CONTENTS

LIST OF TABLES . .
INTRODUCTION .
LTTERATURE REVIEW .

Dental Caries . . . . . . . .
Possible Preventive Measures . . . . .
General Characteristics of Streptococcus Humans
Extracellular Polysaccharides
Glucosyltransferase . . .

 

MATERIALS. . . . . . . . . . . . . . . .

Bacterial Strain .

Growth of Bacterial Cultures .

Preparation of Cultures for Glucosyltransferase Assay
Glucosyltransferase Assay

Glucose Oxidase Assay

METHODS

Growth of Bacterial Cultures . . . . .
Preparation of Glucosyltransferase Sources .
Glucosyltransferase Assay .

Glucose Oxidase Assay .

RESULTS . . . . . . . . . . . .
Addition of Surfactants to Glucosyltransferase Assay
Mixture . . . . . . . .
The Effect of Surfactants on Cell-Associated and
Extracellular Culture Filtrate GTF Activity .
DISCUSSION . . .

LITERATURE CITED . . . . . . . . . . . .

Page
ii

21
21
26
32

Table

1.

LIST OF TABLES

Effect of Glycerol Monolaurin (GML) and Sodium.Laury1
Sulfate (SLS) upon Crude Extracellular Glucosyltrans—
ferase (GTF) of Streptocoocus mmtans 6715 . . . . .

 

Glucosyltransferase Activity in StreptOco¢cuS‘mutans 6715
Cultures Supplemented with Glycerol Monolaurin (GMEI .

 

Glucosyltransferase Activity in Streptococcus mutans 6715
Cultures Supplemented with TWeen’BO or Sodium Lauryl
Sulfate (SLS) . . . . . . . . . . . . . .

 

ii

Page

23

2A

. 25

INTRODUCTION

Fats. which compose 30-AO% of the human diet, have been examined for
their antimicrobial effects and.may be useful as non-toxic prophylactic
agents in the prevention of dental caries. Several studies have demonstrated
that the development of dental caries can be retarded by fats. Lard
(Roseburg, et al., 1939), butterfat (Constant, et al., 195“), corn oil
(Roseburg, et al., 1939), and margarine (wynn, et al., 1960) have all
been shown to inhibit the development of animal caries.

The antimicrobial properties of fatty acids are well known (Bayliss,
1936; Kodicek, 191:9). Kabara et a1. (1972) examined 30 purified fatty
acids and derivatives fer antimicrobial activity against 12 gram-positive

organisms (including Streptococcus mmtans 6715) and 8 gram-negative organisms.

 

Of the saturated fatty acids the average minimal inhibitory concentration

(MIC) decreased from the highest value fer the C6 and C fatty acids to

20
the lowest fer the 012 (lauric acid). Addition of a cis double bond in

the delta 9 position increased the activity of the unsaturated Cl”, 016

and C18 fatty acids. Addition of a second double bond further increased
the bacteriostasis of the C18 fatty acid.

Since lauric acid, C had the greatest antimicrobial activity of

12’
the saturated fatty acids, the effect of esterification of lauric acid
with various alcohols was examined. Lauryl alcohol and lauryl aldehyde

were similar in activity to the free acid. Lauric acid esterified with

2
cholesterol or.methanol showed no inhibition. The diglyceride
(1,3-dilaurin) and triglyceride (trilaurin) were less active than the
fatty acid. The monoglyceride (1—mono—1aurin) was found to be more active
than the free acid.

While screening some 40 natural or synthetic lipophilic compounds
for antimicrobial activity, Kabara et a1. (1977) found that 1—mono-1aurin
was the best antimicrobial, particularly against gramapositive
microorganisms.

Because most cariogenic bacteria are gram-positive microorganisms
and since monolaurin is generally recognized as safe (GRAS) by the
Food and Drug Administration, monolaurin was used in an ‘in_'_ _‘v_iy_<_>_
study to determine its effects upon.the development of dental caries in
OsborneéMendel rats (Kabara, et al., 1979). Rats were fed the modified
high sucrose NIH diet 2000 (Keyes, 1959) containing either 2% CriscoR
(control) or 2% LauricidinB (a 90% pure preparation of the monoglyceride
of lauric acid). The animals were orally inoculated with Streptococcus
EEEEE§.6715 and maintained on the diets fer fbur weeks. At the end of
the experimental period the animals were killed and their teeth were evalua-
ted fOr both microorganisms and dental caries. Due to wide variations
in oral microbial populations among animals, no statistical differences
were observed between the CriscoR and LauricidinR groups. Hewever, a
statistically significant redUction in smooth surface dental caries was
measured in rats fed LauricidinR (Schenmel, et al., 1979).

The antibacterial effect of the surfactant, LauricidinR, may play a
role in the inhibition of dental caries, but other mechanisms may also

contribute to the observed reduction in smooth surface dental caries.

3
One of the inhibiting abilities of surface active compounds has been
shown to be upon the activity of the glucosyltransferase (GTF) enzyme

of many of the cariogenic streptococci, notably, Streptococcus mutans

 

(Christiansen and Kilian, 1975).

The purpose of this investigation is to compare the effects of the
surfactants sodium dodecyl sulfate (SDS) , Tween 80 and glycerol monolaurin
upon the activity and distribution of glucosyltransferase in Streptococcus

mutans 6715.

LITERATURE REVIEW

Dental Caries

 

Dental caries is a unique disease in many respects. It appears
to be a product of civilization because its incidence increases
with the standard of living (Bibby, 1978). 'me disease progresses
with no systemic response, such as inflammation or fever, and no repair
mechanisms are initiated. Development of dental caries is affected by
the character of food, bacteria, saliva and tooth structure.

Almost 100% of the population requires treatment which must be provided
by trained, well-paid technicians who need expensive materials and
equipment . To be effective, preventive measures must demand little
trouble or sacrifice from the public.

In the 1890's, W.D. Miller incubated a tooth in a mixture of
saliva and bread and observed that the salivary bacteria fermented the
carbohydrate and produced sufficient acid to decalcify the tooth
(Miller, 1890) . As a result, Miller proposed that bacteria were
the etiologic agents of dental caries (Harris, 1968). Dental caries
has since been defined as the localized destruction of tooth tissue
by bacterial action (Gibbons and van Houte, 1975) . Germ-free animals
do not develop dental caries (Orland, et al., 1954) and antibiotics
prevent progression of the disease (Shaw, 1959).

The temperature (35-360 C). , availability of nutrients and

5
differences in oxygen tension found in the oral cavity make it a
suitable habitat for many bacterial types. The predominant organisms
identified in dental plaque are facultative streptococci , facultat ive
diphtheroids , peptostreptococci, and small percentages of Veillonella,
Bacteroides, Fusobacteria, Neisseria and Vibrio species (Nolte, 1977).
These organisms can gain access to the oral cavity by water, food,
air or hands . p

The first evidence of dental plaque, and the initiation of dental
disease, is the development of the enamel pellicle which consists of
acidic proteins and glyCOproteins from saliva which adsorb to the
hydroxyapatite of the tooth surface. Microorganisms colonize the
pellicle and their production of extracellular polysaccharides
causes increased plaque volume and “adherence (Frostell and Ericsson,
1978).

Sucrose or other easily fermentable carbohydrates are required to
initiate dental caries (Newbrun, 1967 ; Marthaler, 1967). The cariogenic
streptococci produce the extracellular polysaccharides dextran, mutan
and levan from sucrose by means of the enzymes glucosyl- and fructosyl—
transferase. The ability to form extracellular polysaccharides, and
consequently enhanced plaque production, is considered to be one of the
main explanations for the cariogenicity of Streptococcus mutans
(Fitzgerald, 1976) . An intracellular polysaccharide of Streptococcus
mutans, glycogen, and the extracellular levan can be utilized as sources
of energy, when exogenous sucrose or other simple sugars are absent ,
resulting in the production of acid. While extracellular glucan

(dextran or mutan) may be used as a carbon source, strong acid

6
production is unlikely to occur (Huis in't veld and Dirks, 1978).
Addition of exogenous fermentable sugar to dental plaque results in
rapid acid formation. Acid production by these organisms causes
demineralization of the enamel surface of'the tooth and initiates a

caries lesion.

Possible Preventive Measures

Restriction.of sugar intake, use of sugar substitutes, alteration
of the normal oral flora, immunization and treatments making use of
enzyme inhibitors, antibiotics, hydrolytic enzymes or antiseptics
may all potentially inhibit development of dental caries.

Restriction of sugar from the diet would substantially reduce the
incidence of dental caries; however, it is unlikely that people would
voluntarily alter their diets. An attractive alternative is the use
of sugar substitutes, but relatively few human clinical trials have
been carried out to determine their effects on the incidence of dental
caries.

In order to be effective the sugar substitute has to have
sweetness, palatability, chewability and solubility similar to
conventional sweets (Newbrun and Frostell, 1978). Lycasin, a
hydrogenated potato starch hydrolysate, has been used in human studies
in Sweden and its effect on caries development compared with that of
sucrose. No definitive statistical differences were observed.

Sorbitol has been shown to reduce the incidence of caries
and reduce plaque accumulation in rodents, but this was accompanied by

decreased food intake and weight gains so its effects have been

7
questioned (Newbrun and Frostell, 1978). The addition of sorbitol,
arabinatol, ribitol or mannitol to saliva in the presence of plaque
bacteria all produced decreased plaque pH because of acid production
(Hayes and Rdberts, 1978).

Xylitol has demonstrated very low cariogenicity in human studies
and no pH decrease is Observed when it is added to:mixtures of saliva
and plaque bacteria (Hayes and ROberts, 1978). However, the
reduction in caries may have been due to remineralization of early
lesions once the use of sucrose had been discontinued (Scheinin and
Makinen, 1975). Xylitol usage has resulted in some gastrointestinal
side effects and its cost is approximately ten times greater than that
of sucrose; therefore, the benefits of xylitol usage are in doubt.

Implantation of organisms not normally present in the oral
cavity to alter the microbial balance has been attempted but the
organisms are rapidly eliminated. Tb establish new organisms the
diet and salivary compositions would probably also have to be changed
(Frostell and Ericsson, 1978). Another alternative is to introduce

nonppathogenic Streptococcus mutans into the oral cavity. Dental

 

caries may be decreased due to the decreased cariogenicity of the
competing microorganisms (deStoppelaar, Konig, Plasschaert and van der
Heeven, 1971).

Immunization against dental caries has been attempted but it is
not possible to utilize a specific antibody reaction against the mixed
oral flora. vaccines developed against glucosyltransferase and
fructosyltransferase have failed to protect monkeys from the development

of dental caries (Bowen, Cohen and Colman, 1975) and vaccines against

8
dextrans and mutans have shown no effect.

Enzyme inhibitors have been examined as potential therapeutic
agents; however, most have general activity and are too toxic or they
have specific activities and are too ineffective fer clinical use.

Sodium lauroyl sarcosinate and sodium.dehydroacetate have been used in
toothpastes but evidence fer inhibitory effects on dental caries develOp-
.ment is conflicting (Frostell and Ericsson, 1978). Fluoride inhibits
bacterial growth, polysaccharide production and/or acid production (Hamil-
ton, 1977; Rolla, 1977). High fluoride concentrations are required before
acid production is reduced. Fluoride may reduce plaque formation by
reduction of the free surface energy of the tooth (Bella and Melsen,
1975); however, bacteria may acquire resistance to fluoride by reducing
membrane permeability (Frostell and Ericsson, 1978).

The antibiotics penicillin, aureomycin, terramycin, streptomycin,
bacitracin, subtilin, chloromycetin, concanavalin A, polymixin and
others have been shown to reduce plaque and caries (Loesche, 1975).

The development of drug resistant bacteria and toxic or allergic reactions
of patients become problems upon continued usage.

Hydrolytic enzymes such as pronase, neuraminidases and other
bacterial hydrolases are not effective in dissolving dental plaque.
Proteolytic enzymes such as trypsin and pepsin do not dissolve plaque
and.may damage oral soft tissues. Enzymes which attach the alpha (1,6)—
and a1pha-(l,3)—linkages of extracellular polysaccharides may inhibit
dental plaque fermation, but do not remove established plaque. The
use of enzymes in caries prevention remains open to future investigation

(Frostell and Ericsson, 1978).

After rinsing or brushing with antiseptics the bacterial counts of
the oral cavity are reduced 50%, but the normal flora regenerates in
approximately two hours due to the short generation times (about 20
minutes) of many of the oral streptococci (Loesche, 1975). Antiseptics
are not generally practical for everyday use . Quaternary ammonium com—
pounds reduce plaque flora to 10-30% of the normal population.
Oxidizing agents such as hydrogen peroxide and sodium perborate reduce
plaque accumulation but cannot be used frequently.

The surfactants chlorhexidine, sodium dodecyl sulfate and sodium
deoxycholate have also been shown to reduce plaque, caries and
periodontal disease (Loesche, 1975; Christiansen and Kilian, 1975) but,
again, they cannot be generally used-due to the possible development of
bacterial drug resistance (Frostell and Ericsson, 1978). The patient
may develop toxic or allergic reactions with prolonged usage and taste
sensations are often affected.

General Characteristics of
Streptococcus:mutans

 

 

 

Clarke (1924) originally isolated a group of nonhemolytic

streptococci from carious human teeth which he called Streptococcus

 

mggggys. IMany characteristics of the strains isolated served to distinguish
them from other streptococci. ‘All of the strains fermented mannitol,
sorbitol, inulin, raffinose, salicin, lactose and melibiose. Edwardson
(1967) fbund that these strains farmed dextran from.sucrose, hydrolyzed
esculin, grew in A% NaCl broth and 10% bile agar.

The glucosyl— and fructosyl-transferase enzymes produced by these

organisms utilize the energy contained in the disaccharide bond of sucrose

10

for polymerization of glucan and fructan (Gibbons and van Houte, 1975) .
Dextranase is produced by many strains of Streptococcus mutans (Staat
and Schachtele, 19711). The enzyme may function as a means of using
extracellular soluble dextran during periods of nutritional deprivation
or it may be involved in the format ion of highly branched dextrans by
hydrolyzing soluble dextrans to oligosaccharides which could be
incorporated by glucosyltrans ferase as a branch. Invertase-like
enzymes are also produced by these organisms and may be the main factor

involved in the decomposition of sucrose in dental plaque (Birkhed and

f

Frostell, 1978) .

Streptococcus mutans was found to be serologically heterogeneous.

 

Bratthall (1970) described five type antigens which were designated

a, b, _c_:_, g and 2. Types 3 and b were purified and identified as
polysaccharides present in or on the bacterial cell wall (Mukasa and
Slade, 1973). Type a has major components of rhamnose, galactose and
glucose (Bleiweis, Craig and Zinner, 1971). The serological specificity
of the type a determinant has been related to a terminal diglucose
(Mukasa and Slade, 1973). Type l3 strains of Streptococcus mutans have
a distinct pattern of cell wall carbohydrates. The major sugars present
are rhamnose and galactose (Bleiweis, Craig and Zinner, 1971). Two
type b antigens have been isolated and characterized from Streptococcus
m strain FAl (Mukasa and Slade, 1973). Type b_, II, lacks rhamnose
and contains 39% protein. The antigenic specificity is related to
galactose, rhamnose and protein acting as carriers for the glucose

polymers. Two type 3 antigens were isolated from Streptococcus

 

mutans strain Ingbritt (Linzer, Gill and Slade, 1976). Both were

ll

polysaccharides composed of rhamnose and glucose in a ratio of 2.14:1.
They differed in minor components, phosphorus and protein. The
serological specificity of the 2 antigen is related to a terminal
glucose. The type _d_ antigen is a polysaccharide composed of galactose
and glucose in a 2:1 ratio (Linzer and Slade, 19714). The antigen has
two serologically active sites. One site is specific for group d and a
second site is common to both group d and group 3 strains. The antigenic
specificity of type 31 strains depends on a terminal D-galactose.
The composition of the type _a_ antigen has not been described at this
time, but is similar to type _c_ in that its major carbohydrates are
rhamnose and glucose. Tm new serotypes have been proposed (Perch,
KJems and Ravn, 19714) and designated types _1: and g. Type 3 has the same
carbohydrate pattern as types 3 and 3. Type g has the same carbo-
hydrate pattern as types a and _d_. Both _f and g cross react with their
particular type mates.

As may be expected, Streptococcus mutans strains have been found
to be genetically heterogeneous (Coykendall, 1974) . It was determined
by genetic, antigenic and biochemical tests that the organisms commonly

identified as Strgitococcus mutans from human dental plaque are composed

 

of four different species of streptococci. The characteristic colony
(on media containing sucrose), polymer production and mannitol and
sorbitol fermentation define the "mutans—group" of streptococci
(Coykendall and Lizotte, 1978). The species Streptococcus mutans

has a DNA base content (mole percent Grl-C) ranging from 36-38, does not
produce ammonia from arginine, is resistant to bacitracin and ferments

raffinose. The DNA base content of ‘Streptococcw rattus ranges from

 

12

141-43 and the organism produces ammonia from arginine. Streptococcus
cricetus has a DNA base content of 112—184 and is sensitive to bacitracin.

Streptococcus sobrinus has a DNA base content of All-146 and the organism

 

does not ferment raffinose .

Streptococcus mutans was the predominant Streptococcus mutans—like

 

organism isolated from dental plaque samples tested.
Extracellular Polysaccharides

Human and rodent cariogenic streptococci produce large quantities
of extracellular carbohydrate from sucrose, whereas non-cariogenic
bacteria fail to do so (Gibbons, et al., 1966). Extracellular poly-
saccharide synthesized by cariogenic bacteria was shown to be present
as a component of the matrix of human dental plaque (Gibbons and
Banghart, 1967). At least one fraction of the polysaccharide formed
by the cariogenic streptococcus FA-l was found to be polyglucose with
1-6 and/ or 1-2 hexopyranoside linkages confirming the material to be
dextran (Wood and Critchley , 1966) . 'Ihin layer and paper chromatography
of acid hydrolysates of the polysaccharide produced by streptococcus
strain GS—5 indicate it was primarily a glucose polymer containing
small amounts of fructose and was rapidly hydrolyzed by the dextranase
activity of the culture filtrates of Penicillium funicolosum (N.R.R.L.
1768) (Gibbons, et al., 1966). Dextran (glucan) precipitated by ethanol
from the culture medium of Streptococcus mutans E419 was found to be
highly branched,a glycogen-like structure, with a linear (l, 6)-backbone

and (1,3)- branches (Lewicki, et al., 1971).

13

Water insoluble glucan isolated from culture filtrates of

Streptococcus mutans strain Ingbritt A represented about 25% of the

 

total glucan and contained both alpha-(1,6)- and alpha-(1,3)-linkages.
Water soluble glucan contained principally alpha-( 1, 6 )—1inkages
(Baird, Iongyear and Ellwood, 1973). The absence of virulence
observed in mutants is probably caused by a failure to form the alpha-
(l,3) rich component (Freedman, Birked and Granath, 1978). Four types

of glucan produced by Streptococcus mutans have been isolated, three of

 

which are water soluble. 'Ihe water-insoluble and one of the water-
soluble glucans have molecular weights of 15 x 106, are mostly
alpha-(l,3)-1inked glucose and are resistant to dextranase. rIhe
insoluble glucan may form by aggregation of high molecular weight
water-soluble glucan. The second water-soluble glucan isolated had a
molecular weight ranging from 3 x 105 to 5 x 106, was highly branched,
and resistant to dextranase. The last soluble glucan was dextranase
sensitive, consisted of mostly alpha-(1,6)-linked glucose in a linear
arrangement, and had a molecular weight of 10“ or less (Inoue and

Koga, 1979).

' Glucosyltrans ferase (Dextransucrase)

 

Wood (1967) described the isolation of an enzyme responsible for
the synthesis of dextran from sucrose by streptococcus FA-l.
The activity of the enzyme, dextransucrase, was assayed by estimating
the release of reducing sugar from sucrose using a modification of the
Somogyi method (Marais, et al., 1966). Optimal activity of the enzyme

was between pH 5.2 and 7.0, at 145°C (Carlsson, Newbrun and Krasse, 1969).

1“

No polysaccharide was produced from maltose, lactose, fructose,
melibiose, galactose, glucose, raffinose, trehalose, cellibiose,
melezitose, alpha-methyl glucoside, sucrose monOphosphate or sucrose
diphosphate (Carlsson, et al., 1969; Newbrun and Carlsson, 1969).

Fructose was found to be directly inhibitory for the enzyme
(Gibbons and Nygaard, 1968) . Urea and sodium dodecyl sulfate
completely inhibited activity (Chludzinski, Germaine and Schachtele,
197A). Dextran was found to stimulate activity of the enzyme
(Germaine, Chludzinski and Schachtele, 197A).

Glucosyltransferase activity was almost totally extracellular

when Streptococcus mutans strain 6715 was grown in brain heart infusion

 

broth or trypticase glucose broth. When grown in trypticase soy broth,
A0% of the total glucosyltransferase activity was cell-associated, due
to trace amounts of sucrose in the soy extract (Spinell and Gibbons,
19714) .

The distribution of glucosyltransferase activity (extracellular to
cell-associated) changes depending on the medium used. Media free
of detectable contaminating sucrose favors extracellular enzyme
activity (Janda and Kuramitsu, 1976). Therefore, it has been suggested
that the distribution of glucosyltransferase activity can be regulated
independently of its synthesis (Montville, Cooney and Sinskey, 1977).

The addition of the surfactant Tween 80 to the growth medium
resulted in increased levels of glucosyltransferase. However, Tween 80
did not directly affect the glucosyltransferase activity (Umesaki, Kawai

and Mutai, 1977). Penicillin treatment of Streptococcus mutans

 

enhanced the levels of extracellular glucosyltransferase activity

15

(Janda and Kuramitsu, 1978) as did addition of linoleic or lauric acid

to culture broth (McChesney, et al., 1978). Mono- or divalent

cations increased both total glucosyltransferase activity and production

of water-insoluble glucans (Mukasa, et al., 1979).

MATERIALS

Bacterial Strain

 

Streptoco¢cus mutans 6715, a cariogenic organism first described

 

by Fitzgerald, et al., (1968), was obtained from Dr. Rachel Larson,

National Institute of Dental ResearCh, Bethesda, Maryland, U.S.A.

Growth of Bacterial Cultures

 

The liquid growth.medium fer the bacterial cultures was Trypticase
Soy Broth, pH 7.2, and the solid medium was Trypticase Soy Agar, both
from.Baltimore Biological Laboratories of Cockeysville, Maryland.
The surfactants used were TWeen 80, sodium lauryl sulfate (both from
Sigma Chemical Co., St. Louis, NHssouri) and glycerol monolaurin
(LauricidinR, obtained from Med-Chem Laboratories, Monroe, Michigan).
Thrbidity of growth was measured using a Bausch and.Icmb Spectronic 20.

Preparation of Culturesngr
GlucosyltranSferase Assay

 

 

The 2A-hour bacterial cultures were centrifuged using a Model
HN—S Centringe, International Equipment Company, Needham Heights,

Massachusetts.

16

 

 

17
Glucosyltransferase AsSay

Dextran TL10 was obtained from Pharmacia, Inc. of Uppsala, Sweden.
Sucrose was from.Mallinckrodt Laboratory Chemicals of St. Louis,
Missouri. Sigma Chemical Co., also of St. Louis, was the source for
sodium.acetate, 0.3 N (Ba(OH)2,_ZnSOu, arsenomolybdate and all chemicals

used to prepare both the Somogyi reagent and Nelson's color reagent.

Glucose Oxidase Assay

 

The glucose diagnostic kit NO. 510 from Sigma Chemical Co.,

St. Louis, Missouri, was used for the determination of glucose.

METHODS

Growth of Bacterial CultUres

The organism was grown in trypticase soy broth (BBL, Cockeysville,

Maryland, U.S.A.). When required, Streptococcus mutans 6715 was grown

 

fill

!
in the same medium supplemented with 5, 10, or 15 pg/ml g1ycerol.mono- i-
laurin (LauricidinR, Med—Chem Laboratories, Monroe, Michigan, U.S.A.); i_

10, 100, or 1,000 ug/ml TWeen 80 (polyoxyethylene sorbitan monooleate,
Sigma Chemical, St. Louis, NHssouri, U.S.A.); or 15 ug/ml sodium lauryl
sulfate (Sigma Chemical). The pH of the medium was adjusted to 7.2.
Cultures were incubated at 37°C in the Gas-Pak system (BBL) fer 2A hours.
The cell populations were equalized by adjusting the optical density of

all cultures to 0.5 at 550 nm (106 CFU/ml).

Preparation of Glucosyltransferase Sources

 

The bacterial cells were harvested by centrifugation (10,000 x g).
The culture supernatant was filtered using a 0.22 pm filter (Millipore
Corporation, Bedford, Massachusetts, U.S.A.) and was either used imme-
diately or stored frozen at -3000. The cell-pellet. when required
for use. was subjected to eight cycles of freeze-thaw using dry ice to
break the cells and stored frozen. The cell-pellet was diluted to the same

volume as the corresponding culture supernatant, 50 ml, prior to use.

18

 

19

Glucosyltransferase Assay

 

The glucosyltransferase assay was a modification of the methods
of Christiansen and Kilian (1975) and Chludzinski, Germaine and
Schachtele (197A). The reaction mixtures consisted of 100 mM sucrose,
20 1M dextran T—10, 250 mM sodium acetate buffer (pH 5.5), the
appropriate concentration of surfactant (if required), and the crude
glucosyltransferase preparation; either the culture filtrate
(extracellular) glucosyltransferase or the cell-associated (cell-
pellet) glucosyltransferase. The total volume of the reaction mixture
was 5 ml. The reaction mixtures were incubated in shaker water bath
at 370C. The reactions were stopped by addition of 0.3 N Ba(OH)2
and 5% ZnSOu (Nelson, 19%) and then filtered through Whatman No. 1
filter paper. Three m1 of filtrate were added to 3 ml of Somogyi
reagent (Somogyi, l9ll5; Somogyi, 1952; Marais, et al., 1966); and the
test tubes containing the solutions were placed in a boiling water
bath for 10 minutes, cooled, and 2 ml of Nelson's arsenomolybdate
reagent (Nelson, 19%) were then added. The solutions were diluted to
50 ml in volumetric flasks. Color was allowed to develop, and the
optical density at 750 nm was determined after 30 minutes.

A reagent blank contained 100 mM sucrose, 20 11M dextran T-10 ,

250 mM sodium acetate buffer and boiled (10 minutes) enzyme preparation.
The blank was used to correct for any nonenzymatic activity present in
the reaction mixtures. No growth inhibitor was included in the cell-

associated enzyme assay system.

2O

Glucose Oxidase Assay

 

The glucose oxidase method was used to determine the presence
of free glucose in the enzyme reaction system. The glucose oxidase
assay is specific fer glucose and demonstrated that free glucose is
either not liberated from sucrose, or is liberated at nondetectable

levels under these conditions.

RESULTS

‘ Addition of Surfactants to
GlucosyltranSferase Assay Mixture

Relatively low concentrations (5—15 ugflml) of g1ycerol.monolaurin
(GML) or sodium lauryl sulfate (SLS) added to the glucosyltransferase
(GTF) assay mixture caused a decrease (14—22%) in the crude culture
filtrate GTF activity after a 2 hour interval (Table l). TWeen 80
could not be tested in this manner as a gelatinous precipitate formed
during the Somogyi—Nelson procedure and interfered with subsequent
determination. Using this method, higher concentrations of GML or SLS
gave ambiguous results due to interference caused by their insolubility
at these levels.

The Effect of Surfactants on
Cell—Associated and Extracellular Culture
Filtrate GTF Activity

§_. 133% 6715 was grown in the presence of three surfactants to
determine what effect they would have upon the relative amount of GTF
produced and to determine the percentages of extracellular and cell-
associated GTF. Bacteria were grown for 2“ hours in the presence of
5, 10, 15 ug/ml; and the culture filtrates and cell—pellets were assayed
fer their GTF activity, as described earlier (Table 2). GML supplemented
cultures contained significantly lower levels of culture filtrate

GTF activity compared with controls. Cell-associated activity was

21

22

was variable but similar to control levels.

The bacteria grown in the presence of TWeen 80 or SIS supplemented
media were treated in the same manner (Table 3). Tween 80 added to
the growth medium caused increased levels of GTF activity in both
culture filtrate and cell-associated fractions. Tween 80 at 1,000
ug/ml raised the activity of GTF in whole culture to levels 110% higher
than the control value.

Under these same conditions SLS at 15 ug/ml caused little or no
change in either the amount of culture filtrate or cell-associated

GTF activities.

23

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DISCUSSION

Streptococcus:mutans has been.implicated as a causative agent in

 

the development of smooth surface dental caries in laboratory animals and

humans (Fitzgerald and Jordan, 1968; Fitzgerald, 1968; Guggenheim,

1968; Zinner and Jablon, 1968). Sucrose present in the diet has been

shown to be metabolized by microorganisms on the tooth surface (Krasse,

1965; wood and Critchley, 1966; Gibbons and Banghart, 1967) and can

function as an energy source during bacterial growth or as a substrate for

the glucosyltransferase enzyme(s) (OTTO produced by these organisms

resulting in extracellular glucan formation (Guggenheim, 1968). Only

a small portion of the sucrose is used for glucan synthesis. When

carbohydrates limit growth, it has been shown that less than 15% of

the glucose is polymerized (Robrish and Krichevsky, 1972). However,

the glucans produced have been associated with the attachment of the

microorganism to the tooth surface and the development of dental plaque.
Laboratory animals fed diets containing fats or oils have been

shown to exhibit reduced incidence of dental caries (Constant, et al.,

1954; Roseburg, et al., 1939; Wynn et al., 1960). Schemmel, et a1.

(1979) have demonstrated reduced smooth surface dental caries in rats

fed diets containing 2% glycerol monolaurin. The mechanism of

caries reduction is unknown; however, fats and oils may reduce adherence

of feed or protect tooth enamel from bacterial attack. McChesney, et a1.

26

27

(1978) have postulated that fatty acids may decrease plaque formation,
alter glucan binding by cells, or interfere with bacterial production
of glucosyltransferase. It is possible that the monoglyceride,
glycerol monolaurin, and certain other surfactants may exert their
anticariogenic action in a similar manner.

This report examines two aspects of the effects of surfactants

upon Streptococcus mutans 6715 and the glucosyltransferase produced

 

by this strain of bacteria. The first objective was to measure the
effect of each surfactant upon crude extracellular glucosyltransferase.
The second objective was to determine the relative levels of extra-
cellular and cell-associated glucosyltransferase from bacterial cultures
gown in the presence of these surfactants. Determination of reducing
sugar was accomplished by an adaptation of the Somogyi-Nelson technique
(Nelson, 191m; Somogyi, 191:5; Somogyi, 1952; Marais, et al., 1966) and
was used as an indirect measure of glucan synthesis and, therefore,
glucosyltransferase activity.

The GTF activity was measured by determining the amount of free
fructose released during the GTF reaction with sucrose. In the
reaction between GTF and sucrose, glucose and fructose are formed as
reaction products. While the glucose is polymerized to form a gowing
glucan chain (Wood and Critchley, 1966; Lewicki, et al., 1971), the
fructose becomes available as an energy source for the microorganism
(Gibbons and Banghart, 1967; Robrish and Krichevsky, 1972). The fructose
is measured by an adapted Somogyi—Nelson technique for the determination

of reducing sugars .

28

This indirect method has disadvantages since other enzyme systems
could be present which convert sucrose to its constituent mono-
saccharides, and erroneous conclusions could result. To eliminate
this consideration several control experiments were performed.
Dextranase activity was not observed when the crude enzyme preparation
was incubated with dextran T—lO. When sucrose was incubated with the
crude enzyme preparation, no reducing sugar was detected, indicating
that no invertase activity was present. The absence of fructosyl-
transferase activity was demonstrated by a negative glucose oxidase

test. Streptococcus mutans 6715 has been shown previously to have

 

no (or negligible) fructosyltransferase activity (Montville, Sinskey
and Cooney, 1977; Spinell and Gibbons, ' 197A). Therefore, the activity
measured in the present study is presumed to be glucosyltransferase.
Chludzinski, et al. (197”) reported 95% inhibition of dextran-
sucrase activity by 3.5 mM sodium lauryl sulfate (1,000 ug/ml).
Christiansen and Kilian (1975) examined the effects of some detergents
on the activity of extracellular dextransucrase of Streptococcus mutans
strain Ingbritt and demonstrated the inhibitory effects of chlorhexidine
digluconate , cetyltrimethylammonium bromide, and sodium lauryl sulfate
upon enzyme actitity. These initial results are confirmed and extended
by the findings in the present study. When sodium lauryl sulfate was
added to the GTF assay reaction mixture at 15 ug/ml, approximately 16%
inhibition of enzyme- activity occurred. When added to the GTF assay
mixture at 5, 10, or 15 ug/ml, glycerol monolaurin, which was not
examined previously, inhibited extracellular GTF 114%, 20%, and 22%,

29

respectively. Shklair, et a1. (1981) have also reported the inhibitory
effects of these two compounds, but at much higher concentrations.

They feund SLS and GML inhibit Streptooocous mutans GTF, 95% at 1,000

 

ug/ml and 75% at 5,000 ug/ml, respectively.

TWeen 80 was also tested in the same manner; however, its effect
could not be assessed under our conditions. Upon the addition of
TWeen 80, a gelatinous precipitate fbrmed furing the Somogyi—Nelson
procedure and interfered with the subsequent determination. Others
have shown that this surfactant does not act directly on the enzyme
to stimulate or inhibit catalytic activity (umesaki, Kawai and Mutai,
1977; Shklair, Gaugler and Bruton, 1981).

Where the surfactants, GML, TWeen 80, and SLS, were added to the
culture medium, the effects on the activity and distribution of GTF
between extracellular and cell—associated states were determined.

S. mut____a_n_s_ 6715 was gown for 214 hours in medium supplemented with

0, 5, 10, or 15 ug/ml GML. The GML caused a.marked decrease in
culture filtrate GTF activity, while total activity was only decreased
6-10%.

It was previously shown that media supplemented with the nonionic
detergent TWeen 80 greatly enhance the bacterial synthesis of GTF
(umesaki, et al., 1977; Wittenberger, et al., 1978). This was also
observed in the present study. TWeen 80 resulted in increased
culture filtrate, cell-associated and, therefore, whole culture
activity. At 10, 100, or 1,000 jig/ml the whole culture activity was

11A%, 122%, and 210%, respectively, of the control value.

3O

SLS (15 ug/ml) appears to have no effect upon the production of
GTF in S. w 6715 cultures. The values obtained for culture
filtrate, cell-associated, and whole culture GTF were identical to
control values .

Spinell and Gibbons (19714) described the effect of gowth medium
on the distribution of the GTF in cultures of _S_. ‘m__mi__t_a_n_s_ 6715.
They found that the percent of extracellular GTF varied depending on the
medium in which the organism was gown. When the microorganism was
gown in brain heart infusion or trypticase glucose broth, nearly all
GTF activity was extracellular; but when gown in Trypticase soy broth,
140% of the total GTF activity was cell-associated. This change in
distribution was attributed to trace amounts of sucrose in the soy
extract of Trypticase soy broth. The results in the present study
using Trypticase soy broth indicate approximately 82-81% of the GTF
activity is cell-associated. This percentage was altered by gowth of
the organism in media supplemented with the surfactants GML or Tween 80 .
GML. appears to cause little or no increase in the cell-associated GTF
but a marked decrease in extracellular enzyme. Tween 80, however,
produced the opposite effect. SIS appears to produce no change in GTF
distribution. Higher concentrations of GML (>15 pg/ml) and SIS
(>15 ug/ml) could not be tested for their effects in this manner as
both compounds inhibit gowth of _S_. ‘m__n_i_t_ar_1_§_ 6715 at such levels.

The mechanism involved in altering the distribution of GTF
enzyme was not investigated. However, the incorporation of fatty
acids into bacterial membrane lipid has been shown to cause changes in

the membrane fluidity. This results in an inability to control

31

transport of substances across the membrane (Singer and Nicholson, 1972;
Butcher, et al., 1976; Altenbern, 1977).

Whatever the mechanism involved, all surfactants do not affect the
bacterial cell in the same marmer. Our studies indicate clearly that
Tlveen 80 caused an increase in cellular and extracellular GTF activity.
The glycerol monolaurin did not seem to affect the whole culture level
of GTF but markedly reduced its release. When tested against crude
enzyme preparations, both SIS and GML decreased enzyme activity.

Using the method described, it was not possible to determine the effect
of Tween 80 on GTF activity alone.

In a practical sense glycerol monolaurin may be an effective new
ingredient in a dentifrice or mouthwash because of its antimicrobial
action (Kabara, et a1, 1972; Kabara, et al., 1977), anti-caries effects
(Kabara, et al., 1979; Schemmel, et al., 1979), and its lowering
effect on GTF release and enzymatic activity. Despite these dramatic
biological effects against the microorganism and its end-products,
glycerol monolaurin remains a non-toxic and safe chemical. No other

chemical holds as much promise in the fight against tooth decay.

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32

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3A

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