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This is to certify that the

thesis entitled

HDLECULAR.AND ANTIGENIC CHARACTERIZATION
OF THO UNIQUE EPITOPES FROM THE PHOSPHOLIPASE Bl
MOLECULE OF CANDIDA.ALBICANS

presented by
Eric J. Tarcha

has been accepted towards fulfillment
of the requirements for

14-3- degree in Mal—Laboratory
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6/01 c:lClRCJDateDue.p65—p. 15

 

MOLECULAR AND ANTIGENIC CHARACTERIZATION OF TWO UNIQUE
EPITOPES FROM THE PHOSPHOLIPASE B1 MOLECULE OF CANDIDA
ALBICANS.

By

Eric J. Tarcha

A THESIS

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

MASTERS OF SCIENCE

Clinical Laboratory Sciences Program

2001

ABSTRACT
MOLECULAR AND ANTIGENIC CHARACTERIZATION ON TWO UNIQUE
EPITOPES FROM THE PHOSPHOLIPASE B1 MOLECULE OF CANDIDA
ALBICANS
By
Eric J. Tarcha
Diagnosis of systemic candidiasis, an opportunistic fungal infection
caused by Candida spp. including C. albicans, has been problematic due to the
lack of pathognomonic clinical symptoms, and lack of highly specific and
sensitive diagnostic assays. Recent studies have suggested that the
Phospholipase BI molecule of C. albicans (caPlb1) could be utilized as a
diagnostic marker for candidiasis. In our study two unique epitopes of
caPlb1(Pep1 and Pep2), identified using clustal analysis of caPIb1 with other
fungal Plb1 molecules, were analyzed to determine their fidelity as highly specific
and sensitive molecular markers. A 768 base pair fragment from the caPLB1
gene containing Pep1 and Pep2 was sequenced from five isolates of C. albicans.
There was 100% homology of the deduced amino acid sequences from these
isolates. A bacterial expression system was optimized to express Pep1 from
nested PCR amplicons. lmmunoblotting of Pep1 was perfromed using sera from
a patient with systemic candidiasis. Cross reactivity between the patient sera and
the E. coli proteins in the Pep1 expression samples was observed. Further

immunoblotting experiments, with purified Pep1 and Pep2, should be performed

to fully demonstrate their diagnostic potential.

This thesis is dedicated to my parents, James and Diana Tarcha, whose hard
work and support carried and inspired me throughout my undergraduate and
graduate career at MSU.

ACKNOWLEDGMENTS

I would like to acknowledge and thank the many people who have helped
me complete my collegiate journey through Michigan State University. This long
list includes my advisor, Dr. Leo Mendoza, my graduate committee, the faculty
and staff of Medical Technology Program, all my fellow lab mates, my friend
Roger Herr, and my fiancée Jeannette Porter. All of you have made my trip

down this path worth traveling.

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................... vii
LIST OF FIGURES .................................................................................. viii
INTRODUCTION

Candida albicans ....................................................................................... 1
Candidal infections .................................................................................... 2
Pathogenicity of Candida albicans ............................................................. 4
Immunity to Candida .................................................................................. 6
Incidence of Candidiasis ............................................................................ 8
Treatment of Candidiasis ........................................................................... 8
Diagnosis of Candidiasis ............................................................................ 9
Phospholipase and Candida albicans ..................................................... 11
Research Study ....................................................................................... 16
MATERIALS AND METHODS

Organisms ............................................................................................... 17
DNA isolation ........................................................................................... 17
Polymerase Chain Reaction Amplification of a 768 Base Pair Fragment of
the caPLB1 Gene that Contains the Two Putative Unique Regions ....... 18
Cloning of the 768 Base Pair Fragment of the caPLB1 Gene ................ 20
Long Term Storage of Clones .................................................................. 20
Plasmid DNA Isolation ............................................................................. 21
Sequencing the Clones Containing the 768 Base Pair Fragment of the
caPLB1 Gene ........................................................................................ 21
PCR Amplification of the 117 Base Pair Pep1 and the 81 Base Pair Pep2
Fragments ............................................................................................... 23
Cloning of the 117 Base Pair Pep1 and the 81 Base Pair Pep2 Amplicons
for Expression ......................................................................................... 24
Long-Term Storage, Plasmid DNA Isolation and Sequencing of Pep1 and
Pep2 Clones ........................................................................................... 25
Pilot Expression of the Pep1 Fusion Protein ........................................... 25
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

(SDS-PAGE) ............................................................................................ 27
Coomassie Staining SDS-PA Gels ......................................................... 28
lmmunoblotting ........................................................................................ 28
RESULTS

Amplification of a 768 Base Pair Fragment of the caPLB1 Gene
Containing the Putative Unique Regions, Pep1 and Pep2 ...................... 31
Sequencing of the Clones Containing the 768 caPLB1 Fragment .......... 31

Amplification of the 117 Base Pair Pep1 and 81 Base Pair Pep2

Fragments ............................................................................................... 33
Sequencing of the Clones Containing the Pep1 Fragment ..................... 37
Evaluation of the Pep1 Fusion Protein Expression ................................. 37
lmmunoblotting with Sera from Patients with Systemic Candidiasis ....... 43
DISCUSSION .......................................................................................... 52
BIBLOIGRAPHY ...................................................................................... 59

vi

LIST OF TABLES

Table 1: Primer sets used to amplify the caPLB1 specific regions .......... 19

Table 2: Volumes and concentrations of L-arabinose used for pilot
expression of Pep1 and the positive control ............................................ 27

vii

LIST OF FIGURES

Figure 1: Schematic representation of primer sets used to amplify regions
within the caPLB1 gene.

Primer set one amplifies the 768 bp region that contains the sequences which
encode both the C. abicans-specific peptides Pep1 and Pep2. Primer set two
amplifies a 117 bp fragment that encodes for a 39 amino acid peptide, Pep1.
Primer set three amplifies an 81 bp fragment that encodes a 27 amino acid
peptide, Pep2 .................................................................................................. 19

Figure 2: 768 base pair amplicons of the caPLB1 gene.

A 1.2% agarose gel showing the resulting amplicons from a PCR amplification of
the 768 bp fragments in the five C. albicans isolates. Lane 1 is the A46 isolate,
lane 2 the A55 isolate, lane 3 the A54 isolate, lane 4 the A25 isolate, lanes 5 and
6 the ATCC 36082 strain, lane 7 the fungal control, lanes 8 and 9 the negative
reagent controls and lane L is the 1 Kb DNA Ladder ...................................... 32

Figure 3: Representative sequences of the 768 bp region from each C.
albicans isolate.
The sequences have been aligned and the differences are highlighted ......... 35

Figure 4: Deduced amino acid sequences of the Pep1 and Pep2.
A. The alignment of the deduced amino acid sequences of the Pep1 epitope
in the five isolates of C. albicans used in this study.
B. The alignment of the deduced amino acid sequences of the Pep2 epitope
in the five isolates of C. albicans used in this study ............................. 36

Figure 5: PCR amplification of the Pep1 and Pep2 fragments.

A 1.5% agarose gel showing the resulting amplicons from a PCR amplification of
the Pep1 and Pep2 fragments. Lanes 1 and 2 show the Pep1 amplicons using
plasmid DNA from ATCC 768bp clones 2 and 3 respectively. Lanes 3 and 4
show Pep2 amplicons using plasmid DNA from ATCC 768bp clones 2 and 3
respectively. Lane 5 shows the negative reagent control. Lane L is the 100 bp
PCR Marker .................................................................................................... 38

Figure 6: Alignment of Pep1 clones showing deduced amino acid
sequence of the expression product.

Pep1 clones 2 and 3 are aligned and the insert is in frame with the arms of the
vector. The deduced amino acid sequence for the Pep1 fusion protein is
highlighted ...................................................................................................... 39

viii

Figure 7: Pilot expression of the Pep1 fusion protein.

SDS-PAGE gel stained with Coomassie Blue of 130 RD 6 -Galactosidase
positive expression control (Lane 1), pre-induction expression sample (Lane 2),
Pep1 expression samples with decreasing amount of L-arabinose (Lanes 3-7),
Pep1 pre-induction expression sample (Lanes 8) and Protein Standard (L).
Numbers on the right indicate the molecular weights of the proteins in the
standard. The arrow on the left designates the expression of the 130 kD 6 -
Galactosidase expression control ................................................................... 41

Figure 8: lmmunoblot with anti-V5-HRP antibodies.

lmmunoblot analysis of the 130 kD B -Galactosidase expression control and pre-
expression sample (Lane 1 and 2), Pep1 expression samples with decreasing
amount of L-arabinose and Pep1 pre- induction expression sample (Lanes 3-8),
PositopeTM antibody control protein (Lane 9), and protein standard (Lane 10)
after incubation with anti-V5-HRP antibodies. Numbers on the right indicate the
molecular weight of the proteins in the standard. Note the presence of only one
band in the lane with the PositopeTM antibody control protein ........................ 44

Figure 9: lmmunoblot of 130 kD 8 Galactosidase expression.

lmmunoblot of 130 kD -Galactosidase expression controls (Lanes 1-4), pre-
induction expression samples of the B -Galactosidase expression controls
(Lanes 5-8), and protein standard (Lane L) after incubation with polyclonal anti- [3
~Galactosidase primary antibodies and AP conjugated anti-rabbit lgG secondary
antibodies. Number on the right indicates the molecular weight of protein in
standard. Note the increase of expression of the [3 -Galactosidase molecule in
the post expression samples when compared to the basal levels of expression in
the pre-induction expression samples ............................................................ 46

Figure 10: lmmunoblot of Pep1 expression with anti-V5 primary antibodies
and AP conjugated anti-mouse IgG secondary antibodies.

lmmunoblot analysis of Pep1 post expression samples (Lanes 1-3), Pep1 pre-
induction sample (Lane 4), B -Galactosidase expression control (Lane 5), pre-
induction samples of the B -Galactosidase expression control (Lane 6),
PositopeTM antibody control protein (Lane 7), and protein standard (Lane L) after
incubation with anti-V5 primary antibodies and AP conjugated anti-mouse lgG
secondary antibodies. Numbers on the right indicate the molecular weight of the
proteins in the standard. The asterisk (*) indicates the 8 -Galactosidase
expression control being detected and the double asterisks (**) indicates the
Pep1 fusion protein being detected ................................................................ 48

Figure 11: lmmunoblot of Pep1 samples with sera from a patient with
systemic candidiasis and AP conjugated anti-Human IgG secondary
antibodies.

Immunoblot analysis of TOP1O E. coli cell lysate control (Lane 1), decreasing
amounts of Pep1 expression samples (Lanes 2-4) and protein standard (Lane 5)
after incubation with a 1:1000 dilution of sera from a patient with systemic

candidiasis and AP conjugated anti-Human lgG secondary antibodies. Numbers
on the right indicate the molecular weight of the proteins in the standard ...... 50

INTRODUCTION
Candida albicans

Candidiasis is a fungal infection caused by species in the genus Candida,
primarily Candida albicans (Odds 1988). C. albicans is an opportunistic fungal
pathogen found as a part of the normal animal flora. Hippocrates, in his
”Epidemics”, described thrush, a form of oral candidiasis, as a clinical condition
as early as 400 BC. Throughout history studies have supported Hippocrates’
observation by showing a correlation between candidiasis and previously
debilitated patients (Galen 200 AD, Rosen von Rosenstein 1771 and Undenrvood
1784). Berg was the first to define, in 1846, the relationship between the thrush
fungus and mouth abscesses and hence the pathogenesis of this yeast.

Candida albicans can be isolated from various areas of the human body,
but it is most readily isolated from the alimentary tract and mucocutaneous
regions, with the gastrointestinal tract being the most common source of candidal
infections (Cole et al., 1996, Greenfield, 1991, McCullough et al.,1996, Thomas,
1993). Colonies of C. albicans grown on Sabroud's agar after three days at 25°C
are white to cream colored, smooth, and glistening. After time the colonies tum
waxy and soft, smooth or wrinkled, with or without mycelial fringes due to hyphal
formation. Microscopically, colonies are composed mainly of oval-to-elongate
yeast cells of various sizes that can undergo three different morphogenic
processes, blastospore formation, psuedohyphal formation and hyphal formation
(Kwon-Chung and Bennett, 1992). The parasitic phenotypes of C. albicans can

be demonstrated using hematoxylin and eosin, Gridley, or Gomori-Methenamine

Silver (GMS) stained sections in which they are usually observed as masses of
intra- and extracellular mycelial and yeast elements (Rippon, 1998).
Candidal Infections

Candida albicans is the major opportunistic pathogen in a group of several
pathogenic Candida species known to cause candidiasis mainly in
immunocompromised individuals (Segal and Elad, 1998, McCullough et al.,
1996). Because it is part of the normal human flora, the pathogenesis of this
yeast involves one or more predisposing factors which impair the immune
response to the pathogen (metabolic diseases, HIV/AIDS, immunosuppressive
chemotherapy), produce an imbalance in the flora in favor of the yeasts (use of
broad spectrum antibiotics), or disrupt the cutaneous and mucocutaneous
barriers of infection (surgery and intravenous catheters) (Segal and Elad, 1998).
Appearance of the infection and macroscopic lesions are greatly influenced by
the site of the infection and the competence of the host’s immune system to fight
the pathogen.

Candidal infections can be acute or chronic with nonspecific clinical
manifestations and can be divided into two broad categories: superficial and
systemic (Ashman et al., 1990). Superficial candidal infections are the result of
invasion of the host's outer cutaneous and mucocutaneous layers by the
organism. Topical mucocutaneous and cutaneous lesions are often
characterized as grayish-plaques surrounded by edema, while deeper lesions of
the mucosa, such as those of the esophagus and digestive tract, are observed

as ulcerated areas with the organisms reaching the submucosa and blood

vessels causing hematogenous dissemination (Kwon-Chung and Bennett, 1992,
Odds 1988). Superficial infections can be classified into infections of
mucocutaneous involvement and those of cutaneous involvement.
Mucocutaneous candidal infections include vaginitis, balanitis, alimentary tract
infections, chronic mucocutaneous candidiasis, and oral infections such as
thrush, glossitis, stomatitis, cheilitis, and perleche (Reichart et al., 2000, Thomas,
1993). Cutaneous candidiasis involves infections of the skin and nails mainly
paronychia, onychomycosis, diaper disease, candidal granuloma, and
intertriginous candidiasis (Chapman and Danielle, 1994). T

The spectrum of clinical entities of systemic and deep-seated candidiasis
are variable. The clinical features of systemic candidiasis are non-specific.
Although fever is the most common clinical presentation of candidemia, it is not
pathognomonic (Thomas, 1993). Presentations vary from isolated candidemia
with little or no clinical manifestations to severe septic-like illnesses with multiple
organ failure (Chapman and Daniel, 1994). Almost any organ can be affected,
but disseminated candidiasis is mainly reported in the gastrointestinal tract,
kidneys, eyes, liver and central nervous system (Sobel and Vasquez, 1990).
Candidiasis of the gastrointestinal tract can be localized on the esophageal
mucosa, stomach and/or large and small intestine. The frequency of esophagitis
has increased since the emergence of HIV and AIDS, with 10-30°/o of AIDS
patients that showed symptoms of oral candidiasis having esophagitis (Segal
and Elad, 1998). Infections of the stomach and intestines, which are observed

as white plaques and ulcerations, are common in cancer patients, mainly due to

treatment with antibiotics (switching the flora to favor the fungus) and anti-cancer
chemotherapy (suppression of the immune system). These infections are
usually the source of disseminated candidal infections. Renal candidiasis can
originate from an ascending urinary tract infection or from hematogenously
disseminated organisms (Lundstrom and Sobel, 2001). Intravenous and
intraperitoneal catheterizations are an important source of renal candidal
infections. Ocular infections caused by Candida spp. can be found in both the
outer and inner eye. Inner eye infections are usually the result of hematogenous
dissemination, whereas direct introduction of the fungus into the eye by surgery
or injury can lead to outer eye infections (keratitis). Ocular candidiasis has also
been reported as a secondary infection to chemotherapeutic treatment for other
infections (Segal and Elad, 1998). Hepatosplenic candidiasis is most commonly
seen in individuals with leukemia due to their neutropenic conditions.
Candidiasis of the central nervous system is uncommon, but found primarily in
AIDS patients and pre—tenn infants. This type of infection is usually reported
after disseminated candidiasis, and primarily involves the meninges causing
meningitis (Lipton et al. 1984, Fernandez et al. 2000).
Pathogenicity of Candida albicans

The pathogenicity of C. albicans has been extensively explored. Several
important virulence factors have been identified. These include; 1) the ability of
C. albicans to adhere to host tissues (Calderone et al., 2000, Chafin et al., 1998,
Jong et al., 2001, de Repentigny et al., 2000), 2) the production of proteolytic

and lipolytic enzymes such as the secretory aspartyl proteases and the

phospholipases which seem to facilitate tissue penetration and invasion (Fallon
et al., 1997, Ghannoum, 2000), 3) the yeast to hyphal morphogenesis that
assists tissue penetration (Cutler, 1991, Davis et al., 2000, Phan, Belanger and
Filler, 2000, San-Blas et al., 2000) and 4) immunomodulatory effects of various
fungal constituents that impair the host's immune response (Fidel, Vazquez and
Sobel, 1999, Lui et al., 2001, Noverr et al., 2001). Many breakthroughs in
understanding the pathogenesis of candidiasis have been made in the past ten
years, though it is still not fully understood. It has been found that adhesion
molecules allowing adherence to different types of tissues as well as being able
to bind mucins secreted by mucous and salivary cells, to be initial factors in
dissemination of C. albicans (de Repentigny et al., 2000, Calderone et al., 2000).
These adhesion molecules, which are localized on the fungal cell wall, allow for
the yeasts to bind the host's tissues, colonize, and start the process of invasion.
Invasion of the host tissues is also facilitated by the release of proteolytic
enzymes such as aspartyl proteases that break up the integument. A breach in
any vascularized tissue, the gut mucosal for example, allows for further
dissemination and colonization by C. albicans. As colonization and invasion
proceeds, the yeast to hyphal transition takes place. The production of hyphae,
along with the release of phospholipase molecules, allows for the mechanical
and enzymatic disruption and penetration of host tissues. Hyphal development
also helps to protect the fungus from the host's cellular immune system as the
organism is too large to be phagocytized, as well as by producing. Factors such

as molecular mimicry of host molecules and phenotypic switching are being

studied as important virulence targets for anti-C. albicans drug development
(Soll, 1992, Perez-Martin, Uria, and Johnson, 1999).
Immunity to Candida

The interactions between C. albicans and the host's immune system
during infection are still under investigation. This topic has garnered a
considerable amount of attention and research, yet these studies have been
inconclusive. Studies that have evaluated the immune response during candidal
infections found that there are different immune mechanisms of defense against
the mucocutaneous/cutaneous forms versus the deep-seated forms of
candidiasis, although these differences are not clear-cut (Ashman et al., 1989,
Greenfield, 1992, Fidel, Vazquez, and Sobel, 1999).

Cell-mediated immunity (CMI), mainly the CD4+ T cells, has been shown
to play a large role in the defense of mucocutaneous/cutaneous forms of
candidiasis (Ogawa et al., 1998, Nawrot et al., 2000). This is emphasized by the
fact that individuals who are affected by mucocutaneous/cutaneous candidiasis
are mainly patients with some kind of CMI deficiency (Greenfield, 1992, Segal
and Elad, 1998). HIV/AIDS patients and chronic mucocutaneous candidiasis
patients are examples of people with CMI deficiencies that are highly susceptible
to these types of infections. It has been noted that, although CMI deficient
patients are susceptible to mucocutaneous/cutaneous candidiasis they are rarely
affected by systemic infections unless other predisposing factors are involved
(Ashman and Papadimitriou, 1995). Conversely, systemic candidiasis is usually

observed in patients with severe neutropenia. Therefore, it is evident that

polymorphonuclear cells (PMNs) are important in protection against systemic
candidal infections (Ashman etal., 1989, Greenfield, 1992). Induced
neutropenia in animal models of candidal infections has been shown to increase
susceptibility to systemic candidal infections (Segal and Elad, 1998). CMI
involving CD4+ T-cells has also been reported in the defense against systemic
candidiasis immunity. However, it seems that phagocytic cells play a greater role
in controlling C. albicans systemic infections.

The role of antibody protection in candidal infections remains a
controversial subject. The link that relates a defective CMI or neutropenic state
and an invasive infection is weakened by the fact that these conditions may also
result in a decrease of antibody levels (Casadevall, 1995, De Bemardis et al.,
2000). Therefore, it is not truly understood what role antibody protection plays
during invasive infections. Theories about the mechanisms of protection that
antibodies provide during candidal infections have been suggested. For
instance, anti-candidal antibodies may contribute to the hosts defense by direct
candicidal activity, preventing attachment, providing opsonins that enhance
phagocytosis, binding immunomodulatory molecules, neutralizing extracellular
proteases and phospholipases, and inhibiting yeast-to-hyphae transition
(Casadevall, 1995). Although data concerning the immunity of systemic and
mucocutaneous/cutaneous candidiasis has contributed to our understanding of
the complex interactions between C. albicans and the host, much of it has been

obtained from animal models and in vitro studies. Therefore, there is still much

more to be done to further define and characterize the humoral and cellular
immunity triggered by Candida spp during human infection.
Incidence of Candidiasis
The incidence of systemic candidiasis continues to grow at a dramatic
pace. During a four-year period, Candida species emerged as fourth most
common septicemia and 8% of all nosocomial bloodstream infections in a nation
wide hospital survey (Kalin and Petrini, 1996). More recently, a study reported
that candidal infections were associated with the highest crude mortality when
compared to other nosocomial blood born infections (Edmond et al., 1999).
Mortality rates in immunocompromised patients with systemic candidiasis were
found to be 38-50% even with therapy (Pfaller, 1996, Espinel—Ingroff, 1997).
Treatment of Candidiasis
The treatment of systemic candidiasis caused by C. albicans currently
includes a number of toxic antifungal agents such as amphotericin B,
fluconazole, ketoconazole, miconazole and flucytosine (Thomas, 1993, Hadley
and Karchmer, 1995, Pfaller, 1996, Martin, 1999, Rex at al., 2000). Due to the
difficulty encountered in diagnosing systemic infections, often therapy for
systemic candidiasis is provided based on the patient's clinical symptoms without
a definitive laboratory diagnosis. This practice has led to the increase in anti-

fungal resistance in non-C. albicans species (Rex et al., 2000).

Diagnosis of Candidiasis

Complications in diagnosing systemic candidiasis exist because of the
lack of pathognomonic clinical symptoms presented during infection and the lack
of sensitive and specific diagnostic assays for invasive candidiasis (Venrveij et al.
1998, Vincent et al. 1998, Bucheidt, 2000). High mortality rates and the
administration of empirical anti-fungal chemotherapy are problems clinicians
have had as the result of the difficulties in diagnosing this disease (Matthews,
1996, Maenza and Merz, 1997). Development of fast, highly specific and
sensitive assays for the early and rapid diagnosis of systemic candidiasis could
allow for the early initiation of specific therapy. Early initiation of therapy could
improve the ability of clinicians to manage this important infection, reducing
mortality and anti-fungal resistance.

The current diagnostic methods for disseminated and deep-seated
candidiasis lack the specificity and sensitivity that is required to readily diagnose
this infection (Lew, 1989, Harley and Dummer, 1994, Matthews, 1996). In the
past fifty years, the isolation of Candida in blood culture systems and
demonstration of the pathogen in clinical specimens have been the primary tools
in diagnosing invasive candidiasis (Lew, 1989, Harley and Dummer, 1994).
However, due to their insensitivity, there is concern about the large number of
false-negative results with the blood culture systems (Maenza and Merz 1997,
Segal and Elad 1998, Venlveij et al. 1998). Due to the sub optimal sensitivity of
the culture systems, the development of novel non-culture based methods for

the early specific detection of candidemia are currently under investigation.

Some of the proposed methods to diagnose deep-seated candidiasis involve
non-culture based approaches such as immunodiagnostics, detection of candidal
DNA sequences and detection of candidal metabolites.

At least two types of immunodiagnostic techniques have been explored.
The first system detects the presence of circulating candidal antigens in a
patient's serum or other body fluids and the second is an antibody detection
system against immunoreactive antigens. Latex agglutination, radioimmunoassy
and enzyme-linked immunoassays (ELISA) have been used to detect circulating
cell wall or cytoplasmic candidal antigens such as mannans, aspartyl proteases,
heat shock proteins and enolase enzymes (Bennett, 1987, Bougnoux et al.,
1990, Gutierrez et al., 1993, Harley and Dummer, 1994, Martinez et al., 1998,
VBI'WBIj et al. 1998). Although some of these are promising, issues of low
sensitivity, lack of specificity, and cost effectiveness have deterred their
implementation.

The detection of circulating anti-candidal antibodies has also been
investigated. Anti-candidal enolase antibodies and anti- candidal mannan
antibodies have been detected in sera of patients with invasive candidiasis.
However, low levels of these antibodies in severely immunocompromised
patients have limited their use in diagnostics (Na and Song, 1999, Sendid et al.,
1999). The detection of biochemical metabolites released by Candida spp. has
also been investigated with some success. Metabolites such as D-arabinitol and

1,3-B-D-glucan have shown some promise as putative markers. Most of these

10

metabolite assays are still under investigation (Obayashi et al. 1995, Richardson
and Ellis, 2000).

The use of molecular techniques to detect candidal DNA sequences are
currently being examined as possible tools that could rapidly detect invasive
diseases caused by C. albicans. Amplification by PCR of candidal DNA
sequences and detection of specific sequences with DNA probes in hybridization
assays have been studied (Steffan et al., 1997, Gottfredsson, Cox and Perfect,
1998, Reiss et al., 1998, Wahyuningsih et al., 2000). These assays have proved
to be fast, specific and highly sensitive, although their high sensitivity has posed
a potential disadvantage. Because C. albicans is part of the normal flora in
mammalian hosts, contamination by commensal Candida species could lead to
false positives in these types of assays. In addition, the amplification of
circulating DNA from dead/degraded candidal cells makes these approaches

unsuitable to monitor treatment.

Phospholipase and Candida afiaicans
It was recently found that the phospholipase protein of C. albicans is an
important virulence factor in the pathogenesis of candidiasis and could possible
be used as a diagnostic marker (Ghannoum, 2000). Phospholipases are a
grouping of enzymes that have the capacity to hydrolyze one or more ester
linkages in phospholipids (Streyer, 1995). Because most of these enzymes
target specific ester bonds in their phospholipid substrates, identifying letters

such as A, B, C, and D are used to indicate the specific bond that is cleaved.

11

Phospholipases have been suggested as vinJIence determinants in bacteria,
protozoans as well as fungi (Long-Krug et al., 1985, Saffer and Schwartzman,
1991, Titball, 1993). Pathogens that produce enzymes that hydrolyze
phospholipids and proteins are more likely to be involved in membrane disruption
process during host cell invasion because these molecules target the major
chemical constituents of the host cell envelopes. Organisms that release
phospholipases during invasion of the host’s tissues may rely on the molecules
ability to destroy constituents of the cellular membranes allowing for further
attachment and penetration of the tissues. Hence phospholipases were
suggested as important virulence determinants (Ghannoum, 2000).

To investigate the role of phospholipases as virulence factors during
candidal infections, Ibrahim et al. (1995) compared the abilities of pathogenic
blood isolates versus commensal isolates of C. albicans to produce
phospholipase. They also compared the pathogenicity of clinical isolates that
vary in levels of phospholipase production in a murine model of disseminated
candidiasis. Their studies found that the blood isolates produced significantly
higher levels of phospholipase than the commensal isolates. They also found
that only extracellular phospholipase activity was predictive of mortality among
six other virulence factors examined. Although these findings indicate that
phospholipases might be important in candidal pathogenesis, they do not prove
association between the production of phospholipase and virulence. The fact
that all the isolates were not genetically related does not rule out the possibility

that the difference in pathogenicity is due to factors other than phospholipase

12

activity. To further investigate the role of phospholipase in pathogenicity of
candidiasis, the genes that encode for the phospholipases in C. albicans have
been cloned and sequenced. So far, three genes that encode for C. albicans'
phospholipases named caPLB1, caPLBZ, and caPLD, have been cloned and
sequenced (Mirbod et al., 1995, Leidich et al., 1998, Kanoh et al., 1998,
Sugiyama et al., 1999).

Phospholipase B1 (Plb1) is the dominant phospholipase secreted by C.
albicans (Ghannoum, 2000). This molecule was first purified by Mirbod et al.
(1995) and the gene (caPLB1) was cloned and sequenced by Leidich et al.
(1998). Nucleotide analysis revealed an open reading frame of 1818 bp that
predicted a protein of 605 amino acid residues (Leidich et al., 1998).
Comparisons of this sequence with other phospholipase sequences from
Genbank revealed significant homology. The cloning of caPLB1 allowed for
construction of a caPLBi-deficient mutant strain by gene knockout. These
mutants in turn were used to investigate the role of this molecule in the virulence
of C. albicans. Comparison of the pathogenicity of an isogenic strain pair of C.
albicans, which differ only in the ability to secrete Plb1, was studied in two
different murine models of candidiasis. The virulent properties of the mutant
strain lacking the ability to secrete Plb1 were considerably attenuated, showing
that Plb1 is indeed a putative virulence factor (Leidich et al., 1998). In order to
prove that Plb1 is actively expressed during infection, immunoelectron
microscopic studies were performed using tissues harvested from mice

challenged with C. albicans and Plb1-antiserum (Ghannoum, 2000). This study

13

revealed that Plb1 is expressed and secreted into host tissue during infection
and mainly located at the hyphal tips of the invading cells (Ghannoum, 2000).
Based on this research, the Plb1 molecule has garnered interest as a potential
diagnostic markers for invasive candidal infections.

With evidence that Plb1 is an important virulence factor in candidiasis,
additional studies were performed to investigate the presence of anti-caPlb1 in
patients with and without invasive candidiasis. Western blot analysis was
performed using sera from healthy individuals and patients with invasive
candidiasis against purified Plb1. This study revealed that anti-caPlb1 antibodies
were present in sera obtained from patients with invasive candidiasis, but were
not detectable in sera obtained from healthy volunteers. This study also showed
the immunogenic capabilities of caPlb1 in humans. Preliminary data from
experiments evaluating the levels of anti-Plb1 in patients with invasive candidal
infections from different Candida species and in healthy individuals, showed that
the sera from patients with invasive candidiasis contained significantly higher
levels of anti-Plb1 antibodies in comparisons to the other tested sera. Although
there was a degree of cross reaction between sera from patients with C. albicans
infections and other non-C. albicans species, these results support the idea that
Plb1 could be used as a possible diagnostic marker for candidiasis.

Epitope mapping of immunodominant antigens from infectious organisms,
with the purpose of finding unique peptides (or antibodies against these
peptides) to be used as specific diagnostic markers of infection, has been

investigated with moderate success in microorganisms such as Treponema

14

pal/idum, herpes simplex virus type 2, hepatitis C virus, Streptococcus pyogenes,
Mycobacterium kansasii and Rhodococcus equi (Osborne et al., 1995, Norton,
Heuzenroeder and Manning, 1996, Baughn et al., 1996, Marsden et al., 1998,
Naito et al., 1998, Vanniasinkam, Barton, and Heuzenroder, 2001). Matthews at
al. (1991) first performed epitope mapping of candidal antigens in 1991. This
group developed a limited assay using a rabbit antiserum probe against a
specific epitope on the heat shock 90 protein of C. albicans . Epitope mapping
using clustal analysis was applied to the Plb1 molecule of C. albicans in order to
identify unique immunogenic peptides not present in other fungal Plb1s. It was
proposed that the use of these epitopes or monoclonal antibodies against these
peptides in a serological assay would increase the specificity and sensitivity of
these assays compared to the current serodiagnostic assays. The advantage of
using unique epitopes in these tests lies with the fact that using a monoclonal
antibody to these epitopes may greatly enhance the sensitivity and specificity of
any Plb1 antigen detection assays and the peptides could be used as the
substrate in an ELISA to measure anti-caPlb1 antibody levels in Infected
patients.

The identification of unique epitopes in the Plb1 molecule of C. albicans
was done by aligning the predicted amino acid sequence of candidal Plb1
against those of known fungal Plbs. The alignment was visually inspected for
regions unique to C. albicans. Several conserved regions were found and two
unique variable regions (Pep1 155Met-193Lys, and Pep2 321Glu-Wlle). These

variable regions were present in the same location in all the aligned amino acid

15

sequences indicating that they may be specific for their fungal Plb. The antigenic
potential of these peptides were determined using the ANTHERPROT software
that details factors such as hydrophilicity, hydrophobicity, and solvent
accessibility. Both Pep1 and Pep2 had profiles indicative of potential
antigenicity, which in turn suggests that the host's immune system has access to
these regions.
Research Studv

Based on the uniqueness of Pep1 and Pep2, and their potential as
diagnostic markers, the objectives of this study were threefold.

1.) To demonstrate that the Pep1 and Pep2 amino acid sequences are
conserved among C. albicans isolates.

2.) To optimize a bacterial expression system to express both Pep1 and
Pep2, which ensures an unlimited source of these peptides for later
studies.

3.) To investigate the antigenicity of the expressed peptides by Western

blotting with sera from patients with systemic candidiasis.

16

Materials and Methods

Or anisms

Four clinical isolates and one reference strain of C. albicans were
obtained from Dr. Mahmoud Ghannoum of the Center for Medical Mycology,
Case Western University in Cleveland Ohio. The four clinical isolates used in
this study (A-25, A—46, A-54, and A-55) were isolated from blood cultures in
patients with systemic candidiasis. The reference strain, ATCC #36082, was
originally obtained from the American Type Culture Collection (Rockville, MD).
All isolates were inoculated on Sabouraud agar slants (10 g/L tryptone, 40 glL

glucose, 15 g/L Difco agar) and then incubated and maintained at 25°C.

DNA Isolation

The genomic DNA in all five C. albicans isolates was obtained by a
modified boiling method as per Steffan et al. (1997). Each isolate was streaked
for single colony Isolation on Sabouraud agar plates and incubated at 25°C for
72 hours. For each isolate, a single colony was picked with a sterile toothpick
and resuspended in 50 uL of sterile Tris/EDTA (TE) buffer (10mM Tris-HCI, 1mM
EDTA, pH 8.0) in a 1.5 mL microcentrifuge tube. The tubes were placed in a
boiling water bath for 5 minutes to lyse the cells. After boiling, 250 uL of sterile
TE buffer was used to dilute each sample. The samples were stored at -20°C

unfilneeded.

17

Polymerase Chain Reaction Amplification of a 768 Base Pair Fragment of the

caPLB1 Gene that Contains the Two Putative Unique Regions

Primers were designed to amplify a 768 bp fragment of the caPLB1 gene,
which contained the sequences that encode both Pep1 and Pep2 (Primer Set 1,
Table 1 and Figure 1). These primers were synthesized by IDT, Coralville, IA.
The primers were diluted to a concentration of 5.0 pMol/pL and maintained at -
20°C. Genomic DNA from each of the five C. albicans isolates was used as the
template DNA in amplification reactions. Aspergillus fumigatus DNA was used
as a fungal control to be certain the primers would not amplify a similar sized
product from other fungi. A negative control using ddeO was included. For
each isolate and the controls, a twenty-five uL total volume PCR was set-up in
0.2 mL PCR reaction tubes (Perkin Elmer, Foster City, CA). Each reaction
consisted of: 2.5 pL 10X PCR Gold BufferTM (no MgClg) (Perkin Elmer), 1.5 pL
25mM MgClz solution (Perkin Elmer), 2 uL 10 mM dNTP mix (Perkin Elmer), 5
pmoles of forward primer, 5 pmoles of reverse primer, 0.25 uL AmpliTaq
GoldW(Perkin Elmer), 5.0 pL DNA template, and sterile ddH20 to Q. S. to 25 pL.
PCR amplification was performed with a Perkin Elmer GeneAmp® 9700
therrnocycler with the following cycling parameters: 5 minutes at 95°C followed
by 35 cycles of 1 minute at 94°C, 2 minutes at 50°C, and 3 minutes at 72°C
followed by 1 cycle of 1 minute at 95°C, 2 minutes at 50°C, and 10 minutes at

72°C.

18

The reactions were electrophoresed in a 1.2% agarose TAE (50X: 48.4
g/L tris base, 11.4 mL/L glacial acetic acid, and 20mL/L of 0.5 M EDTA) gel
containing 0.5 ug/ mL of ethidium bromide for 45 minutes at 150V, visualized

and documented on 3 Gel Doc 1000 system (Bio-Rad, Hercules, CA).

Table 1: Primer sets used to amplify the caPLB1 specific regions.

 

 

 

 

 

 

 

 

Primer Region l W‘Forward Primer (5'- 3') Reverse Primer (5'- 3')
Set Amplified - %
i 1 768 bp GGGTTATCAGGTGGATCG TI'GGCCATCTTCTCCACC
' caPLB1
l 2 — Pep1 I ATGTTATCTCAAGG‘ITI’G 'ITTGCTGGCAACTTGATT
; 3 i Pep2 GAAGCGGTGTTGTCTATC TGGATCTACCAAAATATC
' I I

 

 

 

 

 

 

 

 

 

 

768 bp of caPLB‘I

Figure 1: Schematic representation of primer sets used to amplify regions
within the caPLB1 gene.

Primer set one amplifies the 768 bp region that contains the sequences which
encode both the C. abicans-specific peptides Pep1 and Pep2. Primer set two
amplifies a 117 bp fragment that encodes for a 39 amino acid peptide, Pep1.
Primer set three amplifies an 81 bp fragment that encodes a 27 amino acid
peptide, Pep2.

19

Clonianof the 768 Base Pair Fragment of the caPLB1 Gene

The 768 bp amplicon of the caPLB1 gene from each of the five isolates
was cloned separately. The 768 bp fragments were cloned using the TOPO TA
Cloning Kit for Sequencing (lnvitrogen, Carlsbad, CA). The amplicons were
ligated into the pCR 4-TOPO vector as per the manufacturer’s protocol. Briefly,
the amplicons were first ligated into the vector and then transformed into TOP10
E. coli cells. The cells were plated on Luria-Bertani Kanamycin (LBK) agar
(10g/L of NaCl, 109/L tryptone, 5g/L yeast extract, 15 g/L agar, 50 ig/mL
Kanamycin, pH 7.0) for selection of positive clones and incubated overnight at
37°C. Putative positive clones (20 for A-55 and 10 for A-25, A-46, A-54, ATCC
#36082) were picked and plated again on LBK agar plates and incubated
overnight at 37°C. The plates were stored at 4°C until needed for plasmid

isolation and long-term storage.

Long-Term Storage of Clones
Glycerol stocks were made of each clone for long-term storage at -80°C.
Positive clones were streaked onto LBK agar plates for isolation of single
colonies, and incubated overnight at 37°C. A single colony of each clone was
resuspended in a sterile tube containing 2.0 mL of LBK broth and incubated at
37°C with shaking until the cultures reached mid-log phase. Eight hundred and
fifty micro-liters of each culture was mixed with 150 pL of sterile glycerol and

stored at -80°C in a 1.5 mL cryovial.

20

Plasmid DNA Isolation

For each C. albicans isolate, five putative positve clones whose plasmids
contained the 768 bp amplicon were grown in a broth culture and used for
plasmid DNA isolation. Briefly, each clone was streaked onto LBK agar plates
for isolation of single colonies, and grown overnight at 37°C. A single colony
from each clone was resuspended in a sterile tube containing 2.0 mL of LBK
broth and incubated at 37°C with shaking until the cultures reached mid-log
phase. These cultures were centrifuged at 5000 x g for 5 minutes at 25°C to
pellet the cells. The pelleted cells were used for plasmid DNA isolation using the
S.N.A.P. Miniprep Kit (lnvitrogen, Carlsbad, CA) as per manufacturer's
instructions. The resulting purified plasmid DNA was resuspended to a volume

of 60 uL with ddHZO and stored at -20°C.

Sequencing the Clones Containing the 768 Base Pair Fragment of the caPLB1
£310.62
Sequencing of the caPLB 768 bp fragment from the five C. albicans

isolates was done on an ABI Prism 310 Genetic Analyzer (Perkin Elmer, Foster
City, CA) using ABI Prisms' Big DyeTM Terminator Cycle Sequencing Ready
Reaction Kit (Perkin Elmer) to facilitate the sequencing reactions. Three clones
each, were sequenced for isolates A-25, A—46, A-54, and A-55, while 5 clones
were sequenced for ATCC #36082 isolate. All clones were sequenced using
both the M13 fonlvard and M13 reverse universal primers. Sequencing reactions

were prepared using the ABI PRISMTM BigDyeTM Terminator Cycle Sequencing

21

Ready Reaction Kit with AmpliTaq® DNA Polymerase (TRRMXPerkin Elmer).
The sequencing reactions were set-up in 0.2mL MicroAmp® tubes (Perkin
Elmer). To each reaction 4.0 pL of TRRM, 1.0 uL of primer (3.2 pMoI/uL), and
5.0 uL of template plasmid DNA were added. A GeneAmp® 9700 thermal cycler
was programmed to repeat the following thermal profile for 25 cycles: 96°C for
10 seconds, 50°C for 5 seconds, 60 °C for 4 minutes. After the 25th cycle the
reactions were cooled to 4°C and purified for sequencing.

The sequencing reactions were purified using an ethanol precipitation
methodology to remove the excess of dye terrninators in the reactions. Briefly,
each 10 pL reaction was added to a 0.5 mL Genetic Analyzer sample tube
(Perkin Elmer) containing 32 uL of room temperature 95% ethanol and 6 uL of
sterile ddH20. The products were allowed to precipitate at room temperature for
15 minutes. After the precipitation the tubes were centrifuged at full speed for 30
minutes at 25°C. The supernatant was carefully aspirated from each tube and
discarded, taking care not to disturb the pellets. The open tubes were placed in
a vacuum centrifuge and allowed to dry for 5 minutes. The dried pellets were
resuspended in 18uL of template suppression reagent (Perkin Elmer). The
tubes were then heated for 2 minutes at 95°C to denature the double stranded
DNA. Immediately following the 95°C incubation the tubes were placed on ice
for 10 minutes. The tubes were vortexed and centrifuged briefly at full speed at
25°C. The samples were loaded onto an ABI Prism 310 Genetic Analyzer and
run on a 61 cm x 50 um internally uncoated capillary using POP-6 polymer and

310 Genetic Analyzer Buffer with EDTA (Perkin Elmer). The samples were run

22

using the Seq POP6 (1mL) E run module, the DT POP6 {BD Set-Any Primer}
dye set/primer file, and the raw data was analyzed using a matrix that was

constructed on the same 310 for analyzing BigDyeTM terminator chemistry.

PCRflplification of the 117 Base Pair Pep1 and the 81 Base Pair Pepz
Fragments
Nested primers were designed to amplify the 117 bp and 81 bp fragments

of the caPLB1 gene, which encode for the peptides Pep1 and Pep2 respectively
(Primer Set 2 and 3, Table 1 and Figure 1). The primers were designed to allow
the resulting amplicons to be in frame for expression when ligated into the
expression vector. These primers were synthesized by IDT, Coralville, IA. The
primers were diluted to a concentration of 5.0 pMol/pL. Plasmid DNA from the
cloned 768 bp caPLB1 fragment of the ATCC # 36082 isolate, clones #2 and #3,
was used as the template DNA for the amplification reactions. A negative
reagent control using ddeO to replace the template genomic DNA was included.
The reactions contained the same concentrations of reagents as previously
described in the amplification of the 768 bp fragment except that the amount of
template DNA was reduced to 0.5 uL per reaction. The following cycling
parameters were used: 5 minutes at 95°C followed by 25 cycles of 30 seconds at
94°C, 30 seconds at 50°C, and 30 seconds at 72°C followed by 1 cycle of 30

seconds at 94°C, 30 seconds at 50°C, and 10 minutes at 72°C. The reactions

were then electrophoresed in a 1.5% agarose TAE gel containing 0.5 ug/mL of

23

ethidium bromide for 30 minutes at 150V, visualized and documented on 3 Gel

Doc 1000 system.

Cloning of the 117 Base Pair Pam and the 81 Base Pair Per32 Amplicons for
Expression
Both of the 117 bp Pep1 and 81 bp Pep2 amplicons were cloned for the

purpose of expressing the encoded peptides. The PCR parameters and primers
outlined above were used to amplify both fragments from the 768 bp plasmid
DNA of ATCC #36082 clone #2. The resulting amplicons were electrophoresed
in a 1.0% low melting agarose TAE gel containing 0.5 pg/mL of ethidium bromide
for 30 minutes at 150V to gel purify the amplified fragments. The gel was
documented on a Gel Doc 1000 system and the bands of interest were excised
from the gel. The low melting agarose containing the PCR products was
incubated at 65°C until the gel was melted. The mixture was vortexed and
placed in a 37°C heating block and used for the ligation reaction. For cloning
and expression, the pBAD TOPO TA Expression Kit (lnvitrogen, Carlsbad, CA)
was utilized. The pBAD-TOPO 4.1 kb vector utilizes an arabinose promoter for
expression within E. coli. The cloning was as per the manufacturer’s
instructions. Briefly, the amplicons were ligated into the expression vector,
transformed into TOP10 competent E. coli cells, plated on Luria-Bertani
Ampicillin (LBA) agar (109/L of NaCl, 109/L tryptone, 5g/L yeast extract, 15 g/L
agar, 80 ig/mL Ampicillin, pH 7.0) for selection of positive cl ones and incubated

overnight at 37°C. Putative positive clones (20 for each Pep1 and Pep2) were

24

picked and plated again on LBA agar plates and grown overnight at 37°C. The
plates were stored at 4°C until use for plasmid isolation, long-term storage, and
expression. The positive control expression vector containing the sequence for
the B-Galactosidase protein was also transformed into TOP10 cells as per the
instruction manual and used later as a control during the expression

experiments.

Long-Term Storage. Plasmid DNAlsolation and Sequencing of Pep1 and Peg_2_
QM

Long-term storage of the Pep1 and Pep2 clones was done as described
before. However, the LBK agar plates and LBK broth were replaced with LBA
agar plates and LBA broth. Plasmid DNA isolation of 5 putative positive Pep1
and Pep2 clones was done as described before. The Pep1 and Pep2 clones
were sequenced to evaluate if the amplicons were ligated into the vector in the
correct orientation, as well as to verify if they were in frame with the vector for
proper expression. Five clones were sequenced for Pep1 and Pep2 using the

pBAD Fonlvard Primer (lnvitrogen, Carlsbad, CA) as described above.

Pilot Expression of the Pep1 Fusion Protein
A Pep1 clone, whose plasmid was in frame, and a positive control [3-
Galactosidase clone were selected for pilot expression experiments. The clones
were streaked onto LBA agar plates for isolation of single colonies, and

incubated overnight at 37°C. A single colony of the Pep1 clone was used to

25

inoculate 2 mL of LBA broth in a 50 mL conical tube. The same protocol was
used for a positive control colony. The cultures were incubated overnight at
37°C with shaking until the cells reached to an 00600 of ~1.5. Six 50 mL conical
tubes were properly labeled, 1 mL and 10 mL of LBA broth was added to each
tube. Tubes 1-5 were inoculated with 0.1 mL of the overnight Pep1 culture and
the positive control tube was inoculated with 0.1 mL of the ovemight [3-
Galactosidase culture. The tubes were incubated at 37°C with shaking until they
reached mid-log phase (an ODsoo of ~0.5). Meanwhile, four 10-fold serial
dilutions of 20% L-arabinose were made with ddeO using aseptic technique, to
be used later as the stock solutions for induction of expression. Once mid-log
phased was reached, a 1.0 mL aliquot of cells was removed from each tube and
placed in separate 1.5 mL microcentrifuge tubes. These tubes were centrifuged
at full speed for 30 seconds at 25°C to pellet the cells. The supernatant was
removed from each tube and discarded. The pellets were frozen at -20°C until
used for expression analysis. These samples were taken at the zero time point
prior to induction of expression with L-arabinose. The previously prepared stock
solutions of L-arabinose were then added to the six 9.0 mL cultures (Table 2) to
induce expression of the fusion proteins. These cultures were incubated at 37°C
while being rotated for 4 hours. After incubation, 1.0 mL aliquots were taken
again from each culture and treated the same as the zero point samples. These

samples were used to evaluate expression by SDS-PAGE and lmmunoblotting.

26

TABLE 2: Volumes and concentrations of L-arabinose used for pilot expression
of Pep1 and the positive control.

 

 

 

 

 

 

 

 

 

Tube IStock Solution Volume (mL) Final Concentration;
1 0.002% 0.09 : 0.00002%
7 7 2”“ 0.02% 0.09 0.0002%
"3" l ”0.2%" ””“0209” 0.002% '
7775777"i““‘“2070%m77 0.09 0.2% 7
{'fi’aéifdhifl” ”"21’00/3 7?0‘9“” 7, WW”

 

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SOS-PAGE)
To evaluate the expression of the fusion proteins, SDS-PAGE was used

to prepare the samples for Coomassie staining and immunoblotting. Expression
samples (frozen cell pellets) and Positope (lnvitrogen, Carlsbad, CA) blotting
control were thawed at 25°C for 30 minutes and then treated with 100 iL of IX
Laemmli sample buffer (BioRad, Hercules, CA). The samples were then placed
in a boiling water bath for 5 minutes. The samples were allowed to cool at room
temperature. 5 pL of each sample, 10 pL of Positope, and 10 pL of Prestained
SDS-PAGE Broad Range Standard (BioRad) were loaded into a 4%/12%
stacked SOS-Polyacrylamide Gel [Two 12% Resolving Gels (2.5 mL 1.5M Tris-

HCI pH 8.8, 100 uL 10% SDS, 4.0 mL 30% Acrylamide/Bis (29:1) Stock, 50 “L

10% ammonium persulfate, 5.0 uL TEMED, and 3.35 mL ddeO) and Two 4%

27

Stacking Gels (2.5 mL 0.5M Tris-HCI pH 6.8, 100 uL 10% SDS, 1.3 mL 30%
Acrylamide/Bis Stock, 50 uL 10% ammonium persulfate, 10.0 pL TEMED, and
6.1 mL ddH20)] in duplicate. 1X Tris/Gchine/SDS Buffer (BioRad) was used in
the Mini Protean II gel system (BioRad) for the SDS-PAGE. Before the start of
the electrophoresis, two drops of 0.1% Bromophenol Blue was added to the
center chamber. The samples were then electrophoresed at 150V until the dye
front ran off the gel. Note: Some samples were run on 4%/14% stacked SDS-
PAGE gels.
Coomassie Staining SDS-PA Gels

Gels were placed in Coomassie Blue Stain (0.1 g Coomassie blue, 40 mL
methanol, 10 mL acetic acid, 50 mL ddeo) for 45 minutes with gentle shaking.
Gels were then placed in Destain 1 (40 mL methanol, 40 mL acetic acid, 320 mL
ddeO) for 20 minutes 2X with gentle shaking. After the second wash in Destain
1, gels were placed in Destain 2 (80 mL methanol, 40 mL acetic acid, 280 mL
ddH20) with gentle shaking. The gels were stored in Destain 2 until they could
be documented.

lmmunoblotting

After electrophoresis, the 4% stacking gel was removed from the resolving
gel. The SDS-PAGE gels were placed in Transfer Buffer for equilibration (3.03
g/L Tris base, 1.07 g/L glycine, 200 mL/L methanol, pH 8.3 in ddH20) for 10
minutes prior to electro-transfer. The proteins were electro-transferred in
transfer buffer to a 0.45 pm pore Protran Nitrocellulose Membrane (Schleicher

and Schuell, Keene, NH) using a Mini Trans-Blot Cell (BioRad, Hercules, CA) for

28

1 hour at 100V with the cell surrounded by ice. Once transfer was completed,
the gel was coomassie stained to visualize the effectiveness of the transfer and
the membrane was placed in 15 mL of Blocking Buffer [5% nonfat dry milk w/v in
TBST (20 mL/L 0.5M Tris-HCI pH 8.0, 8.8 g/L NaCl, 0.05% Tween 20 in ddH 20)]
and incubated at room temperature for 1 hour with gentle shaking. The
membrane was then washed 2x with TBST for 10 minutes each wash.
Membranes incubated with Anti-V5-HRP antibodies (Invitrogen, Carlsbad, CA)
were placed in a container with 15 mL of Anti-V5-HRP antibody diluted 1:5000 in
dilution buffer (1% nonfat dry milk w/v in TBST) for 1 hour at 25°C with gentle
shaking. Membranes incubated with Anti-B-Galactosidase primary antibodies
(lnvitrogen) were placed in a container with 15 mL of Anti-B-Galactosidase
antibodies diluted 1:5000 in dilution buffer for 1 hour at 25°C with gentle shaking.
Membranes incubated with Anti-V5 (lnvitrogen) primary antibodies were placed
in a container with 15 mL of Anti-V5 antibodies diluted 1:5000 and/or 1:1000 in
dilution buffer for 1 hour at 25°C and/or at 4°C with gentle shaking. Membranes
incubated with sera from patients with systemic candidiasis (Dr. Ghannoum,
Cleveland, OH) were placed in a container with 15 mL of sera from a patient with
systemic candidiasis diluted 1:1000 and/or 1:5000 overnight at 4°C with gentle
shaking. Membranes incubated with Anti-V5 primary antibodies were incubated
with 15 mL of Goat Anti-Mouse lgG Alkaline Phosphatase conjugated secondary
antibodies (Sigma, St. Louis, MO) diluted 1:10000 in dilution buffer for 1 hour at
25°C with gentle shaking. Membranes incubated with B-Galactosidase primary

antibodies were incubated with 15 mL of Donkey Anti-Rabbit lgG Alkaline

29

Phosphatase conjugated secondary antibodies (Promega, Madison, WI) diluted
1:10000 in dilution buffer for 1 hour at 25°C with gentle shaking. Membranes
incubated with sera from patients with systemic candidiasis were incubated with
15 mL of Goat Anti-Human lgG Alkaline Phosphatase conjugated secondary
antibodies (Sigma) diluted 1210000 in dilution buffer for 1 hour at 25°C with
gentle shaking. Horse Radish Peroxidase detection was performed with the
HRP Conjugate Substrate Kit (BioRad). Alkaline Phosphatase detection was
done with the Western Blue Stabilized Substrate for Alkaline Phosphatase

(Promega).

3O

RESULTS

A_mplification of a 768 @se Pair Fragment of the caPLB1 Gene Containing the
Putative Unique Regions. Pfl1 and Pe02
Genomic DNA was isolated from each of the four clinical isolates (A-25,
A-46, A-54, and A-55) and one reference strain (ATCC #36082) of C. albicans.
The genomic DNA from each isolate was used as the template to amplify by
PCR 3 768 bp fragment of the caPLB1 gene that contains the two unique
epitopes Pep1 and Pep2. Aliquots of the amplification reactions were
electrophoresed in an ethidium bromide stained agarose gel to visual the size of
the resulting amplicons (Figure 2). It was observed that amplicons approximately
770 bp in size were amplified from each of the five isolates. The fungal control
reaction (A. fumigatus DNA) showed non-specific amplification of smaller
products. There was no amplification in the negative control. The same
parameters were used for subsequent amplifications of the 768 bp fragments for
cloning purposes.
Sequencing of the Clones Containing the 768 caPLB1 Fragment
To determine variability among the amino acid sequences, the caPLB1
768 bp fragments from the five C. albicans isolates were cloned and then

sequenced. For isolate A-55, clones 1, 3 and 4 were sequenced.

31

12345679L

r I“ 'I ’

 

Figure 2: 768 base pair amplicons of the caPLB1 gene.

A 1.2% agarose gel showing the resulting amplicons from a PCR amplification of
the 768 bp fragments in the five C. albicans isolates. Lane 1 is the A46 isolate,
lane 2 the A55 isolate, lane 3 the A54 isolate, lane 4 the A25 isolate, lanes 5 and
6 the ATCC 36082 strain, lane 7 the fungal control, lanes 8 and 9 the negative
reagent controls and lane L is the 1 Kb DNA Ladder.

32

There was one difference at the nucleic acid between clones 1/3 and clone 4 at
base pair 543 (C to T) and between clones 3/4 and 1 at base pair 639 (T to C).
For isolate A-25, clones 1, 3 and 4 were sequenced. There was one difference
at the nucleic acid level between clones 1/4 and clone 3 at base pair 543 (T to
C). For isolate A-54, clones 1, 2 and 3 were sequenced. There were no
differences at the nucleic acid or amino acid level between these clones. For
isolate A-46, clones 6, 15 and 17 were sequenced. All the sequenced clones
were identical for this isolate. For the ATCC #36082 isolate, clones 1, 2, 3, 5
and 7 were sequenced. There were differences at the nucleic acid level between
clones 1/2/5/7 and clone 3 at base pair 444 (T to C), and at base pair 447 (A to
G). Although there were differences at the nucleic acid level in some of the
clones sequenced, none of the changes affected the predicted amino acid
sequence. An aligned, representative nucleic acid sequence for each isolate is
shown in Figure 3. It was observed that the deduced amino acid sequence was
highly conserved among all the C. albicans isolates in this region(Figure 4).
A_mplification of the 117 Base Pair Pep1 and 81 Base Pair P902 Fragments
Nested primers were designed to amplify the 117 bp Pep1 and 81 bp
Pep2 fragments by PCR for cloning into an expression vector. To verify
amplification of the expected products, aliquots of each reaction were

electrophoresed in an ethidium bromide stained agarose gel. Amplicons

33

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

10
GGGTTATCAG
GGGTTATCAG
GGGTTATCAG
GGGTTATCAG
GGGTTATCAG

70
GATGACATGT
GATGACATGT
GATGACATGT
GATGACATGT
GATGACATGT

130
ATTGAACATC
ATTGAACATC
ATTGAACATC
ATTGAACATC
ATTGAACATC

190
AAAAGAAATG
AAAAGAAATG
AAAAGAAATG
AAAAGAAATG
AAAAGAAATG

250
CCTTTGCTCA
CCTTTGCTCA
CCTTTGCTCA
CCTTTGCTCA
CCTTTGCTCA

310
GCTTCAAACT
GCTTCAAACT
GCTTCAAACT
GCTTCAAACT
GCTTCAAACT

370
CCAGATACTA
CCAGATACTA
CCAGATACTA
CCAGATACTA
CCAGATACTA

430
GGTTCATGGG
GGTTCATGGG
GGTTCATGGG
GGTTCATGGG
GGTTCATGGG

490
GACAATGGTC
GACAATGGTC
GACAATGGTC
GACAATGGTC
GACAATGGTC

GTGGATCGTG
GTGGATCGTG
GTGGATCGTG
GTGGATCGTG
GTGGATCGTG

TATCTCAAGG
TATCTCAAGG
TATCTCAAGG
TATCTCAAGG
TATCTCAAGG

CTATAAAGCA
CTATAAAGCA
CTATAAAGCA
CTATAAAGCA
CTATAAAGCA

CAAATTTTAA
CAAATTTTAA
CAAATTTTAA
CAAATTTTAA
CAAATTTTAA

CAAACACAGA
CAAACACAGA
CAAACACAGA
CAAACACAGA
CAAACACAGA

TCCAAAGCCA
TCCAAAGCCA
TCCAAAGCCA
TCCAAAGCCA
TCCAAAGCCA

CCATTATTAA
CCATTATTAA
CCATTATTAA
CCATTATTAA
CCATTATTAA

ATCCTAGTTT
ATCCTAGTTT
ATCCTAGTTT
ATCCTAGTTT
ATCCTAGTTT

GTCCAACTGG
GTCCAACTGG
GTCCAACTGG
GTCCAACTGG
GTCCAACTGG

GTTAGTTGGA
GTTAGTTGGA
GTTAGTTGGA
GTTAGTTGGA
GTTAGTTGGA

TTTGTGGGAA
TTTGTGGGAA
TTTGTGGGAA
TTTGTGGGAA
TTTGTGGGAA

AGTTGAAGAA
AGTTGAAGAA
AGTTGAAGAA
AGTTGAAGAA
AGTTGAAGAA

CGTTTCTTTA
CGTTTCTTTA
CGTTTCTTTA
CGTTTCTTTA
CGTTTCTTTA

AGACGAAGGT
AGACGAAGGT
AGACGAAGGT
AGACGAAGGT
AGACGAAGGT

CCAAATGCCG
CCAAATGCCG
CCAAATGCCG
CCAAATGCCG
CCAAATGCCG

TCTCAACTCC
TCTCAACTCC
TCTCAACTCC
TCTCAACTCC
TCTCAACTCC

AAATGAATTT
AAATGAATTT
AAATGAATTT
AAACGAGTTT
AAATGAATTT

TAAGTGTTAT
TAAGTGTTAT
TAAGTGTTAT
TAAGTGTTAT
TAAGTGTTAT

34

AGTCTTGCAA
AGTCTTGCAA
AGTCTTGCAA
AGTCTTGCAA
AGTCTTGCAA

CTCACACACT
CTCACACACT
CTCACACACT
CTCACACACT
CTCACACACT

TGGGTTAATG
TGGGTTAATG
TGGGTTAATG
TGGGTTAATG
TGGGTTAATG

ACTGACATAT
ACTGACATAT
ACTGACATAT
ACTGACATAT
ACTGACATAT

GATGCTTATT
GATGCTTATT
GATGCTTATT
GATGCTTATT
GATGCTTATT

TTTCCAATAT
TTTCCAATAT
TTTCCAATAT
TTTCCAATAT
TTTCCAATAT

ACTGTGATTG
ACTGTGATTG
ACTGTGATTG
ACTGTGATTG
ACTGTGATTG

GTTGACACTA
GTTGACACTA
GTTGACACTA
GTTGACACTA
GTTGACACTA

AATGGATTTG
AATGGATTTG
AATGGATTTG
AATGGATTTG
AATGGATTTG

GTAATAATTT
GTAATAATTT
GTAATAATTT
GTAATAATTT
GTAATAATTT

CATTTCTTAG
CATTTCTTAG
CATTTCTTAG
CATTTCTTAG
CATTTCTTAG

TTGGTAATCA
TTGGTAATCA
TTGGTAATCA
TTGGTAATCA
TTGGTAATCA

ATGGAAGACT
ATGGAAGACT
ATGGAAGACT
ATGGAAGACT
ATGGAAGACT

TGTGGTCTGA
TGTGGTCTGA
TGTGGTCTGA
AGTGGTCTGA
AGTGGTCTGA

TGATCAGTGA
TGATCAGTGA
TGATCAGTGA
TGATCAGTGA
TGATCAGTGA

AATTAACTCC
AATTAACTCC
AATTAACTCC
AATTAACTCC
AATTAACTCC

GATATTTGGG
GATATTTGGG
GATATTTGGG
GATATTTGGG
GATATTTGGG

ACAATGCTGG
ACAATGCTGG
ACAATGCTGG
ACAATGCTGG
ACAATGCTGG

60
GAACTCTGTT
GAACTCTGTT
GAACTCTGTT
GAACTCTGTT
GAACTCTGTT

120
TTACTATGGC
TTACTATGGC
TTACTATGGC
TTACTATGGC
TTACTATGGC

180
AGTTGCCAGC
AGTTGCCAGC
AGTTGCCAGC
AGTTGCCAGC
AGTTGCCAGC

240
ATTAAGTTAC
ATTAAGTTAC
ATTAAGTTAC
ATTAAGTTAC
ATTAAGTTAC

300
TGTAACGAGT
TGTAACGAGT
TGTAACGAGT
TGTAACGAGT
TGTAACGAGT

360
TGGTAGAGCT
TGGTAGAGCT
TGGTAGAGCT
TGGTAGAGCT
TGGTAGAGCT

420
ATACGAATTT
ATACGAATTT
ATACGAATTT
ATACGAATTT
ATACGAATTT

480
AACAAAATTA
AACAAAATTA
AACAAAATTA
AACAAAATTA
AACAAAATTA

S40
GTTTTTCATG
GTTTTTCATG
GTTTTTCATG
GTTTTTCATG
GTTTTTCATG

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

A55
A54
A46
ATCC
A25

550
GGCACTTCTT
GGCACTTCTT
GGTACTTCTT
GGTACTTCTT
GGTACTTCTT

610
AGCTTTTTAA
AGCTTTTTAA
AGCTTTTTAA
AGCTTTTTAA
AGCTTTTTAA

670
GACGTGTCTG
GACGTGTCTG
GACGTGTCTG
GACGTGTCTG
GACGTGTCTG

730
TCTCAATCAA
TCTCAATCAA
TCTCAATCAA
TCTCAATCAA
TCTCAATCAA

CTGCTCTTTT
CTGCTCTTTT
CTGCTCTTTT
CTGCTCTTTT
CTGCTCTTTT

AAGATATAAT
AAGATATAAT
AAGATATAAT
AAGATATAAT
AAGATATAAT

CTTACAATCC
CTTACAATCC
CTTACAATCC
CTTACAATCC
CTTACAATCC

AAAATCTTTA
AAAATCTTTA
AAAATCTTTA
AAAATCTTTA
AAAATCTTTA

CAACGAAGCG
CAACGAAGCG
CAACGAAGCG
CAACGAAGCG
CAACGAAGCG

TGATGATATT
TGATGATATT
TGATGATATT
TGATGATATT
TGATGATATT

AAATCCATTT
AAATCCATTT
AAATCCATTT
AAATCCATTT
AAATCCATTT

TTTAGTTGAC
TTTAGTTGAC
TTTAGTTGAC
TTTAGTTGAC
TTTAGTTGAC

GTGTTGTCTA
GTGTTGTCTA
GTGTTGTCTA
GTGTTGTCTA
GTGTTGTCTA

TTGGTAGACC
TTGGTAGATC
TTGGTAGATC
TTGGTAGATC
TTGGTAGATC

TTCAAAAGTT
TTCAAAAGTT
TTCAAAAGTT
TTCAAAAGTT
TTCAAAAGTT

GGTGGAGAAG
GGTGGAGAAG
GGTGGAGAAG
GGTGGAGAAG
GGTGGAGAAG

TCACTGAAGC
TCACTGAAGC
TCACTGAAGC
TCACTGAAGC
TCACTGAAGC

CAATTCTCAA
CAATTCTCAA
CAATTCTCAA
CAATTCTCAA
CAATTCTCAA

CTGGCAGCAA
CTGGCAGCAA
CTGGCAGCAA
CTGGCAGCAA
CTGGCAGCAA

ATGGCCAA
ATGGCCAA
ATGGCCAA
ATGGCCAA
ATGGCCAA

600
TAACATACCT
TAACATACCT
TAACATACCT
TAACATACCT
TAACATACCT

660
GTCCAACATA
GTCCAACATA
GTCCAACATA
GTCCAACATA
GTCCAACATA

720
TACTGCTATT
TACTGCTATT
TACTGCTATT
TACTGCTATT
TACTGCTATT

Figure 3: Representative sequences of the 768 bp region from each C.
albicans isolate.
The sequences have been aligned and the differences are highlighted.

35

1 ll 21 31 41

11ch Pep1 HLSOGLIIIELTHS FL SYYGIEHP IKOVEEUVNVGNOVASK
355 Pep1 HLSOGLIJ'E L'I'HS FL SYYGIEHP IKQVEEUVNVGNQVASK
354 Pep1 HLSUGLUE LTHS FL SYYGIEHP IKQVEEWNVGNQVASK
A46 Pep1 MLSOGLIJE LTHS FL SYYGIEHP IKUVEEUWVGNQVASK
A25 Pep1 HLSUGLIIIE LTHS FL SYYGIEHP IKOVEEUVNVGNUVASK

Consensus mlsqglwelthsflsyygiehpikqveetwnvgnqvask

1 ll 21

ATCC Pep2 EAVLSITEANIPSFLKDIIDDILVDP
1155 Pep2 EAVLSITEANIPSFLKDIIDDILVDP
A54 Pep2 EAVLSITEIINIPSFLKDIIDDILVDP
A46 Pep2 EAVLSITEANIPSFLKDIIDDILVDP
A25 Pep2 EAVLSITEANIPSFLKDIIDDILVDP

Consensus eavlsiteanipsflkdiiddilvdp

Figure 4: Deduced amino acid sequences of the Pep1 and Pep2.
A. The alignment of the deduced amino acid sequences of the Pep1 epitope
in the five isolates of C. albicans used in this study.
B. The alignment of the deduced amino acid sequences of the Pep2 epitope
in the five isolates of C. albicans used in this study.

36

approximately 120 bp in size were observed in the reactions using Pep1 primers
and approximately 80 bp in size in the reactions using Pep2 primers (Figure 5).
There was not amplification in the negative reagent control. The same
parameters were used for subsequent amplifications of the Pep1 and Pep2
fragments for cloning into an expression vector.
Sequencing of the Clones Containing the Pep1 Fragment

Pep1 and Pep2 fragments were cloned into an expression vector and
sequenced to determine if the inserts were in frame for expression. Pep1 clones
2 and 3 were found to be in frame in the expression vector (Figure 6).

Evaluation of the Peg1 Fusion Protein Expression

Pilot expression of the Pep1 fusion protein was evaluated using SDS-
PAGE and lmmunoblotting. Coomassie stained SDS-polyacrylamide gels
revealed the presence of bands at numerous molecular weights in the cell-lysate
expression samples. A prominent band at 130 kD was detected in the B-
Galactosidase positive expression control post expression samples, but it was
not observed in the pre- induction expression samples (Figure 7). There were no
prominent bands at the expected molecular weight of 6-8 kD that might indicate

expression in the Pep1 fusion protein post-expression samples (Figure 7).

37

1 2 3 4 5

 

Figure 5: PCR amplification of the Pep1 and Pep2 fragments.

A 1.5% agarose gel showing the resulting amplicons from a PCR amplification of
the Pep1 and Pep2 fragments. Lanes 1 and 2 show the Pep1 amplicons using
plasmid DNA from ATCC 768bp clones 2 and 3 respectively. Lanes 3 and 4
show Pep2 amplicons using plasmid DNA from ATCC 768bp clones 2 and 3
respectively. Lane 5 shows the negative reagent control. Lane L is the 100 bp
PCR Marker.

38

 

X0356Ww "'I‘IUGCC'I‘AcAAATMT'r'I'W META???” "IA"ACAACCAGATA‘TACXTACCC A'I‘GGGC'IC'I‘G GA'I‘CCGG'DGA

 

ttxttttttt ottttttttt xttxtt ......................................................

TRFFGLEIILFNFKKEIYIP.

-------------------------- 'I'I'IIG 'I'I'I‘AAC'I'I'I'A ACAAGGAGAT ATACA'I‘ACCC AWTCTC GATCCCCTGA
90 100 110 120 130 140 150 I 0

 

 

 

'I‘GACGA'I‘GAC AAGC'TCGCCC 'I'I‘A'TUT'TATC 'ICAAGG'I'T'IC TCGGAAC'ICA CACAC'ICA'I'T 'IC'I'I‘AG'I'I'AC TA'ICGca'T'I‘g
1'70 180 I90 200 210 25:0 2 0 2 0

MK‘ICE‘EAT' mocmwcucu'moc 7‘ ‘ 'I'i‘AATG'I'I‘GG ' ' " TAXI‘CAAF’I‘I‘J GCCAGCAAAA AGOGCGAGC'I'H'I'CAAGG'I'AAG"

 

 

 

.. , . .. . . I . .. . - . I - E .
aACA'ICCTAT AAAGCAAG'I'I" GAAGAATCGG 'I'I'AA'ICT'ICC TAA'ICAAG'I'I‘ GCCAGCAAAA ACK'CCGAGCT 'ICvAAGGTAAG
250 2 0 270 280 290 300 310 320

CCTA'ICCCTA ACCC'IC'ICCT CGG'IC'ICGAT 'ICTACGCG'I‘A CCGG'ICA'ICA 'ICACCA'ICAC CAT'ICvAG'I'T'I‘ AAACGG'IC'IC

:1; . . II ‘ V F T V S
CCTA'ICCCTA ACCC'I‘C'ICCT CGC'IC'ICGAT 'ICTACGCGTA CCGG'ICA'ICA 'ICACCA'ICAC CAVICAG'I'I'I‘ AAACCG'IC‘IC

 

 

 

 

Figure 6: Alignment of Pep1 clones showing deduced amino acid
sequence of the expression product.

Pep1 clones 2 and 3 are aligned and the insert is in frame with the arms of the
vector. The deduced amino acid sequence for the Pep1 fusion protein is
highlighted.

39

Figure 7: Pilot expression of the Pep1 fusion protein.

SDS-PAGE gel stained with Coomassie Blue of 130 kD B-Galactosidase
positive expression control (Lane 1), pre-induction expression sample (Lane 2),
Pep1 expression samples with decreasing amount of L-arabinose (Lanes 3-7),
Pep1 pre-induction expression sample (Lanes 8) and Protein Standard (L).
Numbers on the right indicate the molecular weights of the proteins in the
standard. The arrow on the left designates the expression of the 130 kD B -
Galactosidase expression control.

40

 

  

._ dis :0 we «N... . F...

  

‘l

41

lmmunoblotting with Anti-V5-HRP antibodies (Invitrogen, Carlsbad, CA),
that are specific for the V5 epitope on the expressed fusion proteins, detected
the 50 kD antibody control PositopeTM protein (lnvitrogen) but none of the
expressed proteins (Figure 8). The same results were observed after re-
expression of both Pep1 and the B -Galactosidase positive expression control.

The expression protocol was re-evaluated by expressing and detecting
the products of two clones of the B -Galactosidase positive control. Coomassie
stained SDS-polyacrylamide gels showed again the presence of the 130 kD
band in the post-expression samples. lmmunoblotting using polyclonal primary
anti-bodies against B -Galactosidase detected bands at the same 130 kD
position in all lanes. Stronger bands were always observed in the post-
expression samples (Figure 9).

New, non-conjugated anti-V5 primary antibodies (lnvitrogen, Carlsbad,
CA) were used to detect expressed products in the original Pep1 and Beta-
Galactosidase expression samples. These antibodies detected the 50 kD
antibody control PositopeTM protein (lnvitrogen) as well as the 130 kD B -
Galactosidase expression control. In an effort to detect the Pep1 fusion protein
in the post-expression samples, the primary antibody dilution was reduced to
1:1000, primary antibody incubation time was increased and the amount of
sample was increased. These changes resulted in the detection of the putative
Pep1 fusion protein. lmmunoblotting with these new parameters detected the

PositopeTM 50 kD antibody control protein (lnvitrogen), the 130 kD B -

42

Galactosidase expression control and the ~6.5 kD putative Pep1 fusion protein
(Figure 10).

lmmunoblotting with Sera from Patients with Systemic Candidiasis

In order to investigate the antigenicity of the expressed putative Pep1
fusion protein, post-expression samples and a TOP10 E. coli cell lysate control
were subjected to SDS-PAGE and immunoblotting with sera from patients with
systemic candidiasis. Sera from patient with systemic candidiasis, designated as
CMM 20, was used at a 1:1000 and 1:5000 dilution. Bands were detected at
multiple molecular weights in both the expression samples and the TOP10 E. coli
cell lysate control. A band was detected at the expected 6.5 kD molecular

weight of the Pep1 fusion protein in all samples (Figure 11).

43

Figure 8: lmmunoblot with anti-V5-HRP antibodies.

lmmunoblot analysis of the 130 kD B -Ga|actosidase expression control and pre-
expression sample (Lane 1 and 2), Pep1 expression samples with decreasing
amount of L-arabinose and Pep1 pre- induction expression sample (Lanes 3-8),
PositopeTM antibody control protein (Lane 9), and protein standard (Lane 10)
after incubation with anti-V5-HRP antibodies. Numbers on the right indicate the
molecular weight of the proteins in the standard. Note the presence of only one
band in the lane with the PositopeTM antibody control protein.

44

 

0x 0.»? .II'

A. . He.“
n
. tiyabirfi

3mm N. mm...» Ne

45

Figure 9: lmmunoblot of 130 kD B-Galactosidase expression.

lmmunoblot of 130 kD B-Galactosidase expression controls (Lanes 1-4), pre-
induction expression samples of the B -Ga|actosidase expression controls
(Lanes 5-8), and protein standard (Lane L) after incubation with polyclonal anti- [3
-Galactosidase primary antibodies and AP conjugated anti-rabbit IgG secondary
antibodies. Number on the right indicates the molecular weight of protein in
standard. Note the increase of expression of the B -Galactosidase molecule in
the post expression samples when compared to the basal levels of expression in
the pre-induction expression samples.

46

Ox 9:."

\Iln’tllu

all!!!

.— w h o m V n N_.

47

Figure 10: lmmunoblot of Pep1 expression with anti-V5 primary antibodies
and AP conjugated anti-mouse lgG secondary antibodies.

lmmunoblot analysis of Pep1 post expression samples (Lanes 1-3), Pep1 pre-
induction sample (Lane 4), B -Ga|actosidase expression control (Lane 5), pre-
induction samples of the B Galactosidase expression control (Lane 6),’
PositopeTM antibody control protein (Lane 7), and protein standard (Lane L) after
incubation with anti-V5 primary antibodies and AP conjugated anti-mouse lgG
secondary antibodies. Numbers on the right indicate the molecular weight of the
proteins in the standard. The asterisk (*) indicates the B -Galactosidase
expression control being detected and the double asterisks (**) indicates the
Pep1 fusion protein being detected.

48

0x Qwili

Ox Foul-v

‘IK

.— h m n v n NF

‘Ififi

49

Figure 1 1: lmmunoblot of Pep1 samples with sera from a patient with
systemic candidiasis and AP conjugated anti-Human lgG secondary
antibodies.

lmmunoblot analysis of TOP10 E. coli cell lysate control (Lane 1), decreasing
amounts of Pep1 expression samples (Lanes 2-4) and protein standard (Lane 5)
after incubation with a 1:1000 dilution of sera from a patient with systemic
candidiasis and AP conjugated anti-Human lgG secondary antibodies. Numbers
on the right indicate the molecular weight of the proteins in the standard.

50

m

V

51

DISCUSSION

The need for new, highly specific and sensitive assays to diagnose
disseminated candidiasis has received enormous attention (Lew, 1989,
Matthews, 1996, Verweij etal., 1998, Vincent etal., 1998). Factors that have
contributed to this include the lack of sensitivity and specificity of the current
diagnostic markers, the lack of specific clinical symptoms of systemic
candidiasis, and the high mortality rate associated with this disease.
Investigations into non-culture based methods for diagnosing systemic
candidiasis using traditional method have been difficult. This has led
researchers to explore novel molecular and serological approaches (Vewiej et
al., 1998, Reiss et al., 1998). However, these newly developed assays have
lacked the sensitivity and specificity required in clinical laboratories. Recent
findings suggested that the Phosphlipase B1 molecule of Candida albicans could
be used as a diagnostic marker of systemic candidiasis. Certain advantageous
properties of C. albicans Plb1 make it an attractive marker. These properties
include the fact that Plb1 is a naturally secreted antigenic protein, isolates from
patients with systemic candidiasis secrete higher levels of Plb1 in comparison to
commensal isolates, and the gene and its encoded protein has been purified and
well defined (Ghannoum, 2000).

Epitope mapping has been successfully applied by researchers in the
development of serological diagnostic markers for infectious diseases (Baughn
et al., 1996, Marsden etal., 1998, Naito etal., 1998, Vanniasinkam, Barton, and

Heuzenroder, 2001). We took advantage of this approach to align and map

52

known fungal Plb1s to define unique regions within the molecules. In order to
develop Plb1 as a unique marker for systemic candidiasis, unique regions in the
amino acid sequence of C. albicans Plb1 that were not present in other fungal
Plb1 molecules were identified. These unique epitopes are proposed to be used
in developing new serological tests, to improve old antigen or antibody detection
assays, or their DNA sequences used as the basis for novel nucleic acid based
assays. An advantage in using epitopes specific for C. albicans in a diagnostic
assay is that these peptides could be used to make monoclonal antibodies as
well. These monoclonal antibodies could be later used in a highly specific
antigen detection assay and the peptides in highly specific antibody detection
assays. This could circumvent the possible cross reaction between the whole
Plb1 molecule/anti-Plb1 polyclonal sera and other fungal Plb1s/anti-fungal Plb1
antibodies.

In order for these epitopes to be used in the development of new assays,
several characteristics should be required. These include:
1.) Their amino acid sequences would have to be conserved among all clinical
isolates of C. albicans.
2.) An unlimited source of these peptides must be available.
3.) They must be antigenic to the host immune systems.

In our study we a) found that the amino acid sequences of the unique
epitopes (Pep1 and Pep2) are conserved among five clinical isolates of C.

albicans, b) optimized a bacterial expression system for Pep1 to provide an

53

unlimited source of this peptide, and c) performed initial antigenicity testing of the
Pep1 peptide.

In order to use one of the unique epitopes in diagnostic assays, it was
important to determine the variability of their amino acid sequences among
clinical isolates of Candida albicans. Our strategy was to determine if the amino
acid sequence were too variable in these regions to be used as specific peptides
for C. albicans. If these peptides or monoclonal antibodies to these peptides
were too variable they could not be used to consistently diagnose infections
caused by C. albicans. Our analysis of the amino acid sequence of Pep1 and
Pep2 in four clinical isolates and one reference strain of C. albicans showed that
the 768 base pair region within the caPLB1 gene contains the sequence for the
two unique peptides. Although differences were observed at the nucleic acid
level, none of the base changes resulted in a change in the deduced amino acid
sequence of the peptides. The fact that the amino acid sequences of these
regions were conserved in the five isolates used in this study, indicated to us that
it is likely that these regions would be highly conserved in most clinical isolates of
C. albicans. Therefore, they could be exploited as specific markers.

The possibility of producing unlimited amounts of these peptides is
financially attractive. If these peptides, or monoclonal antibodies made against
these peptides, are to be used in assays in clinical laboratories, a dependable
source of these peptides would be needed. Two potential sources of purified
peptides could be a) commercially synthesized peptides and b) recombinantly

expressed peptides. Commercial synthesis of the peptides is an expensive route

54

to obtaining large amounts of the peptides; therefore we optimized a bacterial
expression system to express Pep1. Once a set of recombinant clones that
express the peptides was established, large amounts of purified peptide could be
obtained relatively inexpensively. In our study both unique regions were
successfully amplified using sets of nested primers and their amplicons were
cloned into an expression vector. These clones were sequenced in order to
determine if there were any mutations were induced during amplification of the
inserts and to determine if they were in frame with the vector for proper
expression. Their characterization showed that the PCR amplification of Pep1
and Pep2 did not result in any mutations and that the inserts were indeed in
frame with the vector.

The optimization of the system to express the Pep1 fusion protein proved
to be a tedious task. Expression of the Pep1 fusion protein was evaluated using
SDS-PAGE and immunoblotting. Initial analysis of Coomassie stained SDS-
PAGE gels of the expression samples indicated that the expression system was
working properly. Although there were no visible bands that were indicative of
Pep1 expression, there was an intense band observed in the expression control
sample, which expressed a 130 kD B-Galactosidase protein. lmmunoblotting of
these expression samples gave contrasting results. Blots reacted with the anti-
V5-HRP antibodies did not indicate that there was expression taking place. After
performing multiple experiments to determine the reason for the contrasting
results it was found that there was a problem with the anti-V5-HRP antibodies.

This was determined after the B -Galactosidase expression control protein was

55

detected using polyclonal anti-B-Galactosidase antibodies. A new anti-V5
antibody was then used in the immunoblotting experiments. This time the
expressed protein was detected.

Initial antigenicity testing was done by immunoblotting with sera from
patients with proven systemic candidiasis. Pep1 expression samples were used
to detect antibodies against Pep1 in the infected patient’s sera. A positive result
would indicate that Pep1 is immunogenic and anti-Pep1 antibodies are present.
The results of these initial experiments were, however, inconclusive. Antibodies
were detected by both, the expression samples and the negative control (E. coli
cell lysate with no vector), at the molecular weight that Pep1 would be observed.
There were also multiple other proteins that reacted with the patient's antibodies
used in the blotting experiment. It could be speculated that the patient
possesses natural antibodies against the E. coli cell lysates used in these
experiments. Unfortunately, there were proteins that reacted at the same
molecular weight as the Pep1 peptides, therefore it cannot be determined if all,
part, or none of the reaction at that particular molecular weight is the result of
anti-Pep1 antibodies reacting with the expressed Pep1 protein.

According to previous software analysis, it was expected that both, Pep1
and Pep2 fusion proteins would be antigenic. This was confirmed when
preliminary data, gathered by our collaborating group in Cleveland, showed that
synthetic Pep1 and Pep2 fragments are indeed antigenic. They showed that
these synthetic peptides were detected by antibodies in patients with systemic

candidiasis when used in an ELISA (Unpublished Data). These results

56

established that patients with systemic candidiasis have antibodies that reacted
against Pep1 and Pep2, and that the titers are significantly higher than those in
healthy control sera.

Further testing needs to be done to evaluate Pep1 and Pep2 as specific
diagnostic markers of systemic candidiasis. For instance, the expressed Pep1
and Pep2 proteins needs to be purified to eliminate other E. coli proteins to
determine the antigenicity of the peptides. Once their expressed products have
been purified and their antigenicity determined, the specificity of the peptides
needs to be evaluated. Evaluation of the specificity for candidiasis could be
accomplished by reacting purified Pep1 and Pep2 with sera from patients with
other diseases including the mycoses. It would be expected that little or no
reaction would be observed if the peptides are indeed highly specific for C.
albicans. Testing should also be done on different patient populations with other
candidal infections to evaluate the utility of the Plb1-specific assay. These
should include patients who are afflicted with mucosal and cutaneous
candidiasis, candidiasis as a result of a neutropenic state, candidiasis from non-
neutropenic diseases such as HIV/AIDS, and the appropriate negative controls
(sera from healthy people). Testing should also be done in order to determine if
anti-fungal therapy affects the detection of anti-Plb1 antibodies or Plb1 antigen.
This is of paramount importance since this assay could also be used to monitor
therapy.

In summary, our study showed that the caPlb1 molecule could be studied

at the molecular level to identify putative unique regions not present in other

57

fungal Plbis. This study found that two previously identified putative unique
regions, Pep1 and Pep2, can be PCR amplified, and cloned, with Pep1 being
expressed. However, additional antigenicity experiments, with the purified Pep1
antigens, should be carried out to fully demonstrate their potential as molecular
markers. Although our antigenicity experiments were not conclusive, we have
developed a system that can be later used to test this approach in other

pathogenic microorganisms.

58

BIBLIOGRAPHY

Ashman RB and JM Papadimitriou. 1995. Production and function of
cytokines in natural and acquired immunity to Candida albicans infections.
Microbiol. Rev. 59(4):646-672.

Ashman RB, JM Papdimitriou, AK Ott, and JR Wannington. 1990. Antigens
and immune response in Candida albicans infections. Immunol. Cell. Biol. 68:1-
13.

Baugn RE, M Demecs, LH Taber, and DM Musher. 1996. Epitope mapping of
b-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidan
(Tpp15) with synthetic peptides. lnffect. Immun. 64(7):2457-2466.

Bennett JE. 1987. Rapid diagnosis of candidiasis and aspergillosis. Rev.
Infect. Dis. 9(2):398-402.

Berg FT. 1846. Om tursk hos barn. LJ Hjerta. Stockholm.

Bougnoux M, C Hall, D Moisscenet D, et al. 1990. Comparison of antibody,
antigen, and metabolite assays for hospitalized patients with disseminated or
peripheral candidiasis. J. Clin. Microbiol. 28(5):905-909.

Buchheidt D, H Sklandny, C Baust, and R Hehlmann. 2000. Systemic
infections with Candida spp. in immunocompromised patients with hematological
malignancies: current serological and molecular diagnostic methods.
Chemotherapy. 46:219-228.

Calderone R, S Suzuki, R Cannon et al. 2000. Candida albicans: adherence,
signaling and virulence. Med. Mycol. 38(Suppl 1):125—137.

Casadevall A. 1995. Antibody immunity and invasive fungal infections. Infect.
Immun. 63(11):4211-4218.

Chaffin WL, JL Lopez-Ribot, M Casanova, D Gozalbo, and JP Martinez.
1998. Cell wall and secreted proteins of Candida albicans: identification,
function, and expression. Microbiol. Molec. Biol. Rev. 62(1):130-180.

Chapman SW, CR Daniell III. 1994. Cutaneous manifestations of fungal
infections. Infect. Dis. Clin. N. Amer. 4:879-910.

Cole GT, AA Halawa, and EJ Anaisse. 1996. The role of the gastrointestinal

tract in the hematogenous candidiasis: from the laboratory to the bedside. Clin.
Infect. Dis. 22(Suppl 2):573-588.

59

Cutler JE. 1991. Putative virulence factors of Candida albicans. Ann. Rev.
Microbiol. 45:187-218.

Davis D, JE Edwards, AP Mitchell, and AS Ibrahim. 2000. Candida albicans
RIM101 pH response pathway is required for host-pathogen interactions. Infect.
Immun. 68(10):5953-5959.

De Bernardis F, G Santont, M Boccanera et al. 2000. Local anticandidal
immune responses in a rat model of vaginal infection by and protection against
Candida albicans. Infect. Immun. 68(6):3297-3304.

De Repemtigny L, F Aumont, K Bernard, and P Belhomeur. 2000.
Characterization of binding Candida albicans to small intestinal mucin and its
role in adherence to mucosal epithelial cells. Infect. Immun. 68(6):3172-3179.

Edmond MB, SE Wallace, DK McClish et al. 1999. Nosocomial bloodstream
infections in United States hospitals: a three-year analysis. Clin. Infect. Dis.
29:239-249.

EspinaI-Ingroff A. 1997. Clinical relevance of antifungal resistance. Infect. Dis.
Clin. N. Amer. 11:929-944.

Fallow K, K Bausch, J Noonan, E Huguenel, and P Tambumi. 1997. Role of
aspartic proteases in disseminated Candida albicans infections in mice. Infect.
Immun. 65(2):551-556.

Fernandez M, EH Moylett, DE Noyola, and CJ Baker. 2000. Candidal
meningitis in neonates: a 10 year review. Clin. Infect. Dis. 31:458-463.

Fidel PL, JA Vazquez, and JD Sobel. 1999. Candida glabrata: review of
epidemiology, pathogenesis, and clinical disease with comparison to Candida
albicans. Clin. Microbiol. Rev. 12(1):80-96.

Galen. 130 - ca200. De remedies parabilibus I - III. In: Kuhn CG (ed) 1965.
Opera Omlia. Geoge Olms, Hildersheim.

Ghannoum M. 2000. Potential role of phospholipases in virulence and fungal
pathogenesis. Clin. Microbiol. Rev. 13(1):122-143.

Gottfredsson M, GM Cox, and JR Perfect. 1998. Molecular methods for
epidemiological and diagnostic studies of fungal infections. Pathol. 30:405—418.

Greenfield RA. 1992. Host defense system interactions with Candida. J. Med.
Vet. Mycol. 30289-104.

60

Gutierrez J, C Maroto, G Piedrola et al. 1993. Circulating candidal antigens
and antibodies; useful markers of candidemia. J. Clin. Microbiol. 31(9):2550-
2552.

Hadley S and AW Karchnur. 1995. Fungal infections in solid organ transplant
recipients. Infect. Dis. Clin. N. Amer. 9(4):1045-1074.

Harley WB and SJ Drummer. 1994. Diagnosis of disseminated candidiasis by
detection of antigenaemia: a critical review. Infect. Dis. Clin. Practice. 3:168-
1 72.

Hippocrates. ca 460-377 BC. Epidemics, book 3. Translated F Adams. 1939.
Williams and Wilkens, Baltimore.

Ibrahim AS, F Mirbod, SG Faller et al. 1995. Evidence implicating
phospholipase as a virulence factor of Candida albicans. Infect. Immun.
63(5):1993-1998.

Jong AY, MF Stins, S Huang, SHM Chen, and KS Kim. 2001. Transversal of
Candida albicans across the human blood-brain barrier in vitro. Infect. Immun.
69(7):4536-4544.

Kalin M and B Petrini. 1996. Clinical and laboratory diagnosis of invasive
candida infections in neutropenic patients. Med. Oncol. 13:223-231.

Kanoh H, S Nakashima, Y Zhao et al. 1998. Molecular cloning of a gene
encoding phospholipase D from the pathogenic and dimorphic fungus Candida
albicans. Biochim. Biophys. Acta. 1398:359-364.

Kwon Chung KJ and JE Bennett. 1992. Medical Mycology. Lea and Febiger,
Philadelphia. 280-336.

Leidich SD, AS Ibrahim, Y Fue et al. 1998. Cloning and disruption of caPLB1,
a phospholipase B gene involved in the pathogenicity of Candida albicans. J.
Biological Chem. 273(40):26078-26086.

Lew MA. 1989. Diagnosis of systemic Candida infections. Ann. Rev. Med.
40:87-97.

Lipton SA, WF Hickey, and JH Morris. 1984. Candidal infections in the central
nervous system. Am. J. Med. 76:101-108.

Long-Krug S, K Fischer, R Hysmith, and J Ravdin. Phospholipase A

enzymes of Entamoeba histolytica: description and subcellular localization. J.
Infect. Dis. 152:536-541.

61

Lui L, K Kang, M Takatara, KD Cooper, and MA Ghannoum. 2001. Hyphae
and yeasts of Candida albicans differentially regulate interieukin-12 production
by human blood monocytes: inhibitory role of Candida albicans germination.
Infect. Immun. 69(7):4695-4697.

Lundstrom T and J Sobel. 2001. Nosocomial candiduria: a review. Clin.
Infect. Dis. 32:1602-1607.

Maenza JR and WG Merz. 1997. Candidemia: epidemiology and laboratory
detection. Infect. Dis. Clin. Practice. 6:83-88.

Marsden HS, K MacAulay, J Murry, and IW Smith Indentification of an
immunodominant sequential epitope in glycoprotein G of herpes simplex virus
type 2 that is useful for serotype-specific diagnosis. J. Med. Virol. 56:79-84.

Martin MV. 1999. The use of fluconazole and intraconazole in the treatment of
Candida albicans infections: a review. J. Antimicrob. Chemother. 44: 429-437.

Martinez JP, ML Gil, J Lopez-Ribot, and WL Chaffin. 1998. Serological
response to cell wall mannoproteins and proteins of Candida albicans. Clin.
Microbiol. Rev. 11(1):121-141.

Matthews RC. 1996. Comparative assessment of the detection of candidal
antigens as a diagnostic tool. J. Med. Vet. Mycol. 34:1-10.

Matthews R, JP Bumie, and W Lee. 1991. The application of epitope mapping
in the development of a new serological test for systemic candidosis. J.
Immunol. Methods. 143273-79.

McCullough MJ, BC Ross, and PC Reade. 1996. Candida albicans: a review
of its history, taxonomy, virulence attributes, and methods of strain
differentiation. Int. J. Oral Maxillofac. Surg. 25:136-144.

Mirbod F, M Banno, MA Ghannoum et al. 1995. Purification and
characterization of lysophosphlolipase—transacylase (h-LPTA) from a highly
virulent strain of Candida albicans. Biochim. Biophys. Acta. 1257:181-188.

Na B and C Song. 1999. Use of monoclonal antibody in diagnosis of
candidiasis caused by Candida albicans: detection of circulating aspartyl
proteinase antigen. Clin. Diag. Lab. Immunol. 6(6):924-929.

Naito M, N Ohara, S Matsumoto, and T Yamada. 1998. Immunological

characterization of alpha antigen of Mycobacterium kansasii: b-cell epitope
mapping. Scand. J. Immunol. 48:73-78.

62

Nawrot J, K Grzybek-Hryncewicz, J Zielska, A Czamy and J Podwinska.
2000. The stud of cell-mediated immune response in recurrent vulvovaginal
candidiasis. FEMS Immunol. Med. Microlbiol. 29:89-94.

Narrow RE, M Huezenroeder, and PA Manning. 1996. Antigenic epitope
mapping of the M24 protein of Streptococcus pyogenes: implications for
serodiagnosis of rheumatic fever. FEMS Immunol. Med. Microlbiol. 16:267-271.

Noverr M, SM Phare, GB Toews, MJ Coffey and GB Huffnagle. 2001.
Pathogenic yeasts Cryptococcus neofonnans and Candida albicans produce
immunomodulatory prostaglandins. lnfec. Immun. 69(5):2957-29-63.

Obayashi T, M Yoshida, T Mori et al. 1995. Plasma 1-3-Beta-glucan
measurements in diagnosis of invasive deep mycosis and fungal febrile
episodes. Lancet. 345:17-20.

Odds FC. 1988. Candida and Candidosis. Bailliere Tindall W. B. Saunders.
London.

Ogawa H, RC Summerbell, KV Clemons et al. 1998. Dermatophytes and host
defense in cutaneous mycoses. Med. Mycol. 36(Suppl 1):166-173.

Perez-Martin J, JA Uria, and AD Johnson. 1999. Phenotypic switching in
Candida albicans is controlled by a SIR2 gene. EMBOJ. 18(9):2580—2592.

Pfaller MA. 1996. Nosocomial candidiasis: emerging species, reservoirs, and
modes of transmission. Clin. Infect. Dis. 22(Suppl 2):885-94.

Phan QT, PH Belanger, and SG Filler. 2000. Role of hyphal formation in
interactions of Candida albicans with endothelial cells. Infect. Immun.
68(6):3485-3490.

Reichart PA, LP Samaranayke, and HP Philipsen. 2000. Pathology and
clinical correlates in oral candidiasis and its variants: a review. Oral Diseases.
6:85-91.

Reiss E, K Tanaka, G Bruker et al. 1998. Molecular diagnosis and
epidemiology of fungal infections. Med. Mycol. 36(Suppl 1): 249-257.

Rex JH, TJ Walsh, JD Sobel et al. 2000. Practice guidelines for the treatment
of candidiasis. Clin. Infect. Dis. 30:662-678.

Richardson M and M Ellis. 2000. Symposium on invasive fungal infections:
clinical and laboratory diagnosis. Hospital Med. 61(9):610-614.

63

Rippon JW. 1998. Candidiasis and the pathogenic yeasts. Medical Mycology.
3rd edn. WB Saunders, Philadelphia. 532-581.

Rosa C, S Osborne, F Goretto et al. 1995. Epitope mapping of the NS4 and
N85 gene products of the hepatitis C virus and the use of chimeric NS4-NS5
synthetic peptides for serodiagnosis. J. Virological Meth. 55:219-232.

Rosen von Rosenstein N. 1771. Underrattelse om barrns sjukdomer och
deras botemedal. Wenneberg and Nordstrom, Stockholm.

Saffer LD and S Schwartzman. 1991. A soluble phospholipase of Toxoplasma
gondii associated with host cell penetration. J. Protozool. 38:454-460.

Sanblas G, LR Travasso, BC Fries et al. 2000. Fungal morphogenesis and
virulence. Med. Mycol. 38(Suppl 1):79-86.

Segal E and D Elad. 1998. Candida species and Blastoschizomyces capitus.
Top/e y and Wilson's Microbiology and Microbial Infections vol. 4 Medical
Mycology. Arnold, New York. 423-460.

Sendid B, M Tarbouret, JL Parot et al. 1999. New enzyme immunoassays for
sensitive detection of circulating Candida albicans mannan and antimannan
antibodies: useful combined test for diagnosis of systemic candidiasis. J. Clin.
Microbiol. 37(5):1510-1517.

Sobel JD and J Vasquez. 1990. Candidemia and systemic candidiasis.
Semin. Respir. Infect. 5:123-137.

Soll DR. 1992. High frequency switching in Candida albicans. CMR. 5(2):183-
203.

Steffan P, JA Vasquez, D Boikov et al. 1997. Identification if Candida species
by randomly amplified polymorphic DNA fingerprinting on colony lysates. J. Clin.
Microbiol. 35(8):2031-2039.

Streyer L. 1995. Biochemistry. 4th edn. WH Freeman and Company. New
York. 689-690.

Sugiyama Y, F Nakashima, F Mirbod et al. 1999. Molecular cloning of a
second phospholipase B gene from Candida albicans. Med. Mycol. 37:61-67.

Thomas I. 1993. Superficial and deep candidosis. Inter. J. Derrnatol. 11:778-
783.

Titball RW. 1993. Bacterial Phospholipases. Clin. Microbiol. Rev. 57:347-366.

64

Underwood M. 1784. A treatise on the disease of children. P 43. J Matthews,
London.

Verwiej PE, D Poulan, T Obayashi et al. 1998. Current trends in the detection
of antigenaemia, metabolites, and cell wall markers for diagnosing and
therapeutic monitoring of fungal infections. Med. Mycol. 36(Suppl 1):146-155.

Vincent JL, E Anassie, H Bruining et al. 1998. Epidemiology, diagnosis, and
treatment of systemic Candida infections in surgical patients under intensive
care. Intensive Care Med. 24:206-216.

Wahyuningsih R, H Freisleben, H Sonntag, and P Schnitzler. 2000. Sinple

and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of
invasive candidiasis. J. Clin. Microbiol. 38(8):3016-3021.

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