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This is to certify that the

thesis entitled

Investigation of adherence of
Candida albicans, other Candida spp.,
and Torulopsis glabrata to human buccal

epithelial cells in vitro

 

 

presented by

Laurey Robin Hanselman

' has been accepted towards fulfillment
of the requirements for

M.S. degreein Botany and Plant
Pathology

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INVESTIGATION OF ADHERENCE OF CANDIDA ALQLQAflg

OTHER QANDIQA SPP., AND IOBQLQPSIS QLABRAIA TO
HUMAN BUCCAL EPITHELIAL CELLS IN VIIBO

BY

Laurey Robin Hanselman

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

<3 4BR?

(J 5.5:.

ABSTRACT
INVESTIGATION OF ADHERENCE OF QANQIDA ALEIQANg

OTHER QAEDIDA SPP., AND IQEQLQB§I§ QLAEBAIA T0
HUMAN BUCCAL EPITHELIAL CELLS In £1139

BY

Laurey Robin Hanselman

Adherence of gangigg albiggng to human buccal
epithelial cells was proportional to germ tube formation
capability, and greater than Igrulgpsis glaprata, Q.
tropicalis. 5L. pseudgtrenicalis. 5L» bargesilesis. anui Q-
gtgllatgigga. Adherence of formalized and viable yeast for
gaugguy; spp. and I. gugbggta isolates was similar.
Inhibition of adherence was greatest for FHA treatment of
organisms, although citric acid, con-A, and a-D-methyl-
mannopyranoside treatment decreased adherence equally.
Greatest decreases in adherence were seen for Q. albiggng
isolates, although changes in adherence did not correlate
with germ tube formation capability. Compared to g.
albicans, variations in adherence with treatment inhibition
methods for non-g. .glbigang, organisms ‘were consistent,
suggesting that differences in cell wall structure, mannan
concentration, and adhesin characteristics exist. little
variation in adherence to individual or pooled human buccal
epithelial cells for treated or viable organisms was seen.

This is the first comparison of inhibition of adherence in
non-Q, albicans candida spp. and I. glabrata isolates.

This thesis is dedicated to my father,
Melvin C. Hanselman,

whose love, determination to live,
and extraordinary courage
have given me inspiration,
compassion, and strength
of spirit to endure.

p.
p.
p.

ACKNOWIBDGEMENTS

I would like to express my most sincere gratitude to
Dr. Alvin L. Rogers, Dr. Karen Klomparens and Dr. Everett
8. Beneke, my graduate committee. Their support, advice,
and critical reading of this manuscript are appreciated.
To Dr. Rogers, my major professor, mentor, and friend, I
would like to express my most sincere appreciation for
giving me the opportunity to learn, for introducing me to
the world of Medical Mycology and Microbiology, and for
allowing me to develop my teaching ability. I would also
like to thank Dr. Larry Winberry for his assistance, wealth
of knowledge and friendship, and for allowing me the
latitude to discover and begin to develop my research
capabilities and inquisitive mind.

To my "family" I would like to express my gratitude
and appreciation. Their love, understanding, encourage-
ment, and friendship have given me guidance and emotional
support in difficult times. My most sincere thanks to
Donna Duberg for' her* genuine Ikindness, generosity, and
warmth, and to Linda Kimball for comforting me and
listening always.

Finally, I would like to thank the real "teachers" in

my education thus far who have instilled in me a sense of

iv

purpose, dedication to excellence, and love of learning.
These people have demonstrated and demanded persistence,
dedication, enthusiasm, and honesty when pursuing goals.
The following quote emphasizes these ideals and has given
me a sense of determination in difficult times. I dedicate
this quote to those who have touched my life, enhanced my
education, and especially to those very special people who

have been a source of strength, courage, and love.

It may be we shall touch
happy isles,

And see the great Achilles,
whom we knew,

And though much is taken,
much abides:

We are not now that strength
which in olden days moved
earth and heaven,
But that which we are, we are . . . .

We are one equal temper of
heroic hearts, made weary
by time and fate,

But strong in will to strive,
to seek, and to find,

But NEVER to yield.

Closing stanza of
Tennyson's poem
"Ulysses."

TABLE OF CONTENTS

Eage
LIST OF TABLES O O O O O O O O O O O O O O O O O O O O Vii

LIST OF FIGURES O O O O O O O O O O O O O O O O O O O Viii
INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O 1

LITERATURE REVIEW . . .

HOST DEFENSE . . . . . . . . . . . . . . . . . . 11
VIRULENCE. . . . . . . . . . . . . . . . . . . . 15
CELL WALL STRUCTURE. . . . . . . . . . . . . . . 19
BACTERIAL ADHERENCE. . . . . . . . . . . . . . . 21
ADHERENCE OF CANDIDA SPECIES . . . . . . . . . . 23
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 34
BUCCAL MUCOSAL CELLS . . . . . . . . . . . . . . 34
ORGANISMS. . . . . . . . . . . . . . . . . . . . 34
ADHERENCE ASSAY. . . . . . . . . . . . . . . . . 35
TREATMENT METHODS. . . . . . . . . . . . . . . . 36
STATISTICS . . . . . . . . . . . . . . . . . . . 38

RESULTS 0 O O O O O O O I O O O O O O O O O O O O O O 3 9
DISCUSSION 0 O O O O O O O O O O O O O I O O O O O O O 8 6

APPENDIX A-I. . 102

APPENDIX A-II . . . . . . . . . . . . . . . . . . . . 103
APPENDIX A-III. . . . . . . . . . . . . . . . . . . . 104
APPENDIX A-IV . . . . . . . . . . . . . . . . . . . . 105
APPENDIX B. . . . . . . . . . . . . . . . . . . . . . 106
APPENDIX C. . . . . . . . . . . . . . . . . . . . . . 109

LITEMWRE CITED 0 O C C O C O O O O O O O O O O O O O 115

vi

1321:

LIST OF TABLES

gag e

Comparison of the effect of treatment methods
including formalization of the yeast, cell wall
mannan removal, mannan inhibition, and incubation
of a complementary sugar of mannan in the
adherence assay, on the adherence of isolates

of Candida albicans, other Candida Sppu and
morglopsis glabrata to human buccal epithelial

cells. 0 O O O O O O O O O O O O O O O O O O 0 80
Comparison of the remaining relative percentage

of adhering yeast to human buccal epithelial
cells after various treatment methods. . . . . . . 84

vii

LIST OF FIGURES

Rage

Comparison of adherence of Candida
isolates of differing germ tube formation
capacity to human buccal epithelial cells. . . . 41

Comparison of adherence of clinical isolates

organdidasp. andmnusisglahratat

human buccal epithelial cells. . . . . . . . . . 44

Comparison of the effect of different treatment
methods on adherence of Candida

isolate MSU-I to six different human buccal
epithelial cell samples. . . . . . . . . . . . . 46

Comparison of the effect of different treatment
methods on adherence of Candida albicans

isolate 10-16-35F to six different human buccal
epithelial cell samples. . . . . . . . . . . . . 48

Comparison of the effect of different treatment
methods on adherence of Candida

isolate ONC-1134 to six different human buccal
epithelial cell samples. . . . . . . . . . . . . 50

Comparison of the effect of different treatment
methods on adherence of Candida albicans

isolate DA-05900 to six different human buccal
epithelial cell samples. . . . . . . . . . . . . 52

Comparison of the effect of different treatment
methods on adherence of gangigg_a1higgn§

isolate UCLA to six different human buccal
epithelial cell samples. . . . . . . . . . . . . 54

Comparison of the effect of different treatment

methods on adherence of Candida albicang
isolate 1840 to six different human buccal

epithelial cell samples. . . . . . . . . . . . . 56

viii

10

11

12

13

14

15

16

LIST OF FIGURES, CONT'D.

Comparison of the effect of different treatment

methods on adherence of

isolate #1
epithelial

Comparison of the effect of different treatment

methods on adherence of

isolate #3
epithelial

Comparison of the effect of different treatment

adherence of Qandida atallatdidaa
to four different human buccal epithelial

methods on

cell samples . . . . .

Comparison of the effect of different treatment

methods on adherence of Qdflflifid
to four different human buccal epithelial cell

samples. . . . . . . .

Comparison of the effect of different treatment
methods on adherence of gandida trdpidalis to

four different human buccal epithelial cell

samples. . . . . . . .

Comparison of the effect of different treatment

methods on adherence of dandida
to four different human buccal epithelial cell

samples. . . . . . . .

Comparison of the effects of treatment methods

on adherence of dandida albidana strains to
human buccal epithelial cells.

Comparison of the effects of treatment methods
on adherence of isolates of Idzdldpaig
(non-Q albinans) to human

and Qandida spp-

buccal epithelial cells.

ix

glanrata

to six different human buccal
cell samples.

to six different human buccal
cell samples.

2232

58

60

62

64

66

68

78

82

INTRODUCTION

Microbial infections have plagued humankind throughout
history (Sherris, 1984). Adhesion of microorganisms to
epithelial and mucosal surfaces is considered to be an
initial and essential stage in the colonization and
infection of the host (Beachey et al., 1981). The
adherence mechanism utilized by various bacteria in causing
disease has been determined, but for other pathogenic
microorganisms such as Qagdjda albidafi and other dandida
sp. , the precise mechanism of adherence has not been
established. Candidiasis is a primary or secondary
infection involving a member of the genus gaadida and is
considered to be opportunistic since these organisms are
usually,” saprophytic endogenous species within the human
host (Odds, 1979) . Superficial candidiasis is among the
most common of all infections ,ViFh- “oralflvand vaginal
candidiasis as its most prevalent forms (Hurley, 1980).
Although adherence of Q. Ma to mucosal surfaces is
neither necessary for its survival or for its growth (Odds,
1979), it represents an initial step of the more complex
phenomenon of colonization of tissue surfaces for which
effective mechanisms of cellular attachment have been
developed (Tronchin et al., 1984). Recent research
suggests that the mannan component of the cell wall of Q.
dlhiEdDE is primarily responsible for the adherence of this
organism to epithelial cell surfaces of mucous membranes

1

(Poulain et al., 1978), although the adherence mechanism of
other Qaadida sp. is not known. Understanding the
adherence mechanism utilized by these organisms in the
establishment of disease should lead to a better
understanding of candidiasis and to possible prevention of
infection.

The objectives of the following experiments were to
investigate the differences in adhesion ability of d.
albidana, isolates of differing germ tube formation
capability to human buccal epithelial cells, to determine
the differences in adherence of different species of
Qandida and Idzdldpaia glabgaga to human buccal epithelial
cells using viable yeast, to determine the effect of
formalization of yeast cells on adherence, to compare the
influence of two mannan inhibitors on adhesion of these
organisms to epithelial cells, to determine the effect of
decreased cell wall mannan on adherence ability, and to ’
determine the effect of a complementary sugar of mannan on
adhesion in the adherence assay. Experiments were designed
to compare the adherence of various gandida sp. to buccal
mucosal cells in a group of normal or candidal free
individuals using viable and fermalized yeast cell
preparations .. Other' experiments involved. the ‘use of

methods designed to competitively inhibit adherence of
Qandida sp. and I. glam to buccal epithelial cells,

based on the concept of mannan-mediated adherence. In
these studies, yeast cells were subjected to treatment
methods which permitted binding of the cell wall mannan
ligands through the use of the mannose-specific lectin
concanavalin-A (Sandin and Rogers, 1982), incubation of the
adherence mixtures with a-D-methylmannopyranoside (Sandin
et al., 1982), and ‘treatment of’ the yeast cells with
filamentous hemagglutinin (FHA), a protein adhesin of
fidzdatalla paztdgaig which preferentially binds mannan
(Sato and Sato, 1984). In another experiment, cell wall
mannan of yeast isolates was decreased using a citric acid
extraction technique (Ramada et al., 1981). These
organisms were then used in adherence assays to determine
the effect of decreased cell wall mannan on adherence of
different daddida sp. and T. gldDIQEd to buccal epithelial
cells. In addition, the relationship of germ tube
formation to percent adherence of d. albidand yeast

isolates to buccal epithelial cells was examined.

LITERATURE REVIEW

Candidiasis is one of the oldest diseases known to
humankind. Thrush, an oral Qan_d_i_da infection, was
described in debilitated patients by Hippocrates in his
Epidemics (Odds, 1979; Rippon, 1982). Candidiasis {is a
primary or secondary infection caused by members of the
genus Qapdjda, asporogenous yeasts that reproduce by
forming blastoconidia and do not possess a capsule. They
may form hyphae or pseudohyphae, and belong to the form-
class Deuteromyc‘etes and the form-family Cryptococcaceae
(Rippon, 1982). Within the genus Qandida,q,Q.,__albicans .is.
the most pathogenicsmember, although, other members of the
genus can cause disease--(flurley, 1980). The seven most
virulent species of Qandida include Q. deiQdflé. Q.
Mia. 9%. W (Qandida) guardian.
Q. M, and Q. parapsildsig (Hurley, 1980). In a study

done by Klotz et al., 1983, adherence to vascular

W

 

endothelium was ’greatest with Q. albidand and Q.
M,“ less with Q. msai, and least with Q.
nananaileaia. Q- neaadntraniaalia. and T- 91.1% ta. In in
m studies, adherence of Q. albidads and Q. W
to mucosal epithelialwcells exceeded that of other Qaadida
sp. (Rotrosen et al., 1986). In another study these two
organisms togefih§¥-_,_.,caused approximately 80% of the
candidiasis [cases seen clinically while Q. W and
I. glabraga comprised 10-15% of isolates (Hopfer, 1985).

4

These studies reflect known virulence of different Candida
sp. in 2119.

Candida a;_1dan_ is considered to be normal flora on
alimentary tract mucosal surfaces (Odds, 1979; Rippon,

1982). It is an opportunistic yeast that leads a

~.__- --._. “"' '

saprophytic‘ existence “inn the gastrointestinal and
urogenital tracts of 30 -50% of normal humans (Miles at al.,
1977). It is believed that this organism never leaves the
host spontaneously during life once it becomes part of the

fecal flora (Miles et al., 1977). Thirty to fifty percent

.9. a- .i ‘7

I _._.r-

of normal humans harbor Q. albicans in their gut (Miles et
al., 1977), 39% of normal females have it in their vagina
(Carter et al., 1959), 30% have it in their oral cavity
(Baum et al., 1960) and in 46% of normal individuals, it is
present on perianal skin (Recio and DeLeon, 1957). In
addition, recovery of Q. aim from the human body
increases two-fold in hospital patient populations as
compared to healthy individuals, with the exCeption of
skin, which remains the same regardless of the health of
the individual (Odds, 1979). .

Since Q. albidana is normal flora in the human host,
any invasion is considered to be opportunistic. Qandig
amigans differs from other Qandida sp. in that it is
rarely found outside the natural animal host (Drake and

Maiback, 1973) or as. a :normal resident of skin flora

(Greshem and Whittle, 1961). Factors predisposing an
individual to candidiasis include extremes in age,
physiological changes such as endocrine dysfunction or
diabetes, prolonged administration tof‘ antibiotics,
immunosuppressive (agents or’ corticosteroids, ”generalized
debility» such as in end-stage terminal cancer or
immunodeficiency _states, and iatrogenic mechanical or
trauma induced barrier breaks (Rippon, 1982).

Candidiasis presents clinically as a wide spectrum of
disease states including superficial colonization,
mucocutaneous involvement, systemic infections, and
allergies (Rippon, 1982), with the most common
manifestations including oral and vaginal candidiasis
(Hurley, 1980). Oral candidiasis is produced by overgrowth
and colonization of the tongue, soft palate, buccal mucosa
or other oral surfaces with Q. QM or other Qanm
sp. Its distribution is discrete, confluent, or patchy and
presents as a white to gray pseudomembrane covering the
area. Oral candidiasis is a common problem that presents as
a chronic recurring infection which can be a reservoir for
severe, spreading, localized, or systemic disease in the
compromised host (Epstein et al., 1984). Oral candidiasis
of the neonate has been reported as high as 18%, but the
average is approximately 4%, with infection resulting from

passage through the birth canal (Rippon, 1982). In

newborns, a small amount of the organism in the oral cavity
is a prelude to clinical thrush (Rippon, 1982). Since the
normal resident flora of the gastrointestinal tract is
unestablished at birth, if Q. alnidana is not present by
the third day of life, clinical thrush rarely developes
(Rippon, 1982). Studies have shown that antepartum
treatment with clotrimazole lowers the vaginal Qandida
contamination and consequent thrush in newborn infants
(Rippon, 1982).

Oral candidiasis can be found in patients of all ages
(Rippon, 1982), and in a study of adherence to human buccal
epithelial cells, it was found that adherence was the same
in normal adults and children, suggesting that a stable
cell receptor system is present which is not age dependent
(Cox, 1983). In addition, adherence of Q. m to
buccal epithelial cells from full-term infants and healthy
school age children was lower than adherence to epithelial
cells from premature infants until 5 days of age (Cox,
1986) .

In adults, predisposing factors to oral candidiasis
include avitaminosis (riboflavin deficiency), diabetes
complications, polyendocrine disturbance, neoplasia,
steroids, antibiotics, and other drugs (Rippon, 1982). For
example, chronic oropharyngeal candidiasis is recognized as

a complication of inhaling steroids such as beclomethasone

diproprionate for treatment of respiratory disease (Rippon,
1982). Increased candidal adherence to buccal epithelial
cells during antibiotic therapy is considered to be an
important factori explaining the increased incidence of
Qandida colonization in patients receiving antibiotics and
the persistence of the organism which then permits disease
development (Cox, 1983).

Denture stomatitis is recognized as the most common
form of oral candidiasis (Odds, 1979) and is characterized
by erosive and painful lesions of the oropharynx (Rippon,
1982). Acrylic is considered to be a possible reservoir
for Q. albi cans in denture stomatitis (McCourtie and
Douglas, 1981), and. the. presence of glucose and other
sugars besides sucrose increases adherence of Q. albicans
to acrylic (McCourtie et al., 1981). This is important
because there is increased salivary glucose in diabetic
patients and those undergoing antibiotic or steroid therapy
(Knight and Fletcher, 1971), predisposing these patients to
candidiasis (Odds, 1979). In addition, dental plaque is a
contributing factor in denture stomatitis and is known to
be increased by high dietary sucrose (Ramada and Slade,
1980). Although ELIQELQQQQQES nngana, the major component
of dental plaque (Hamada and Slade, 1980) has no
significant effect on candidal adhesion to epithelial cells

and HeLa cells when incubated in adherence assays,

Straw aaliyaniua and We ni'aiar
constituents of normal oral flora, decrease candidal
adhesion, and incubation with saliva increases candidal
adhesion to epithelial cells and HeLa cells, suggesting
that multiple factors are involved in the pathogenesis of
denture stomatitis (Samaranayake and MacFarlane, 1982).
Vaginal candidiasis is also a common disease caused by
Qandida sp. and presents as a thick, yellow, milky
discharge with patches of a gray-white pseudomembrane on
the vaginal mucosa (Rippon,1982). Conditions which
predispose the patient to vaginal candidiasis include
diabetes, antibiotic therapy, oral contraceptives and
pregnancy, especially during the third trimester (Rippon,
1982). Infections do occur in nonpregnant patients prior
to menstruation (Rippon, 1982). For example, in one study
30% of vaginitis in pregnant females and 18% in
nonpregnant females with vaginal discharge was caused by Q.
alnidana (Holti, 1966). In in 212:2 studies, researchers
found an increase in Q. alnidana adherence in situations
where there was an increase in the number of intermediate
epithelial cells. In addition, adherence of Q. alnidans
isolates from patients with vaginitis was significantly
higher than that of isolates of asymptomatic carriers
(Segal, et al., 1984). Scanning electron microscopy

studies have shown non-uniform distribution of adhering

10

microorganisms with diminished adherence in areas of
active mitosis and proliferation, and increased adherence
to mature flat cells often in the process of desquamation
(Sobel, et al., 1982). Local defense factors present in
the vaginal area include a preventative coating of vaginal
cells with lactobacilli which decrease the pH of the
environment and decrease the number of adhering Q. albicans
(Sobel et al., 1981). The pH of the vagina is increased
and is optimum for candidal vaginitis during and after
menses because lactobacilli are less prevalent in the
vagina at that time (Mead, 1974). In addition, recurrent
vaginitis is a common problem in patients and it is
postulated that the continuous presence of Q. albicans in
the gastrointestinal tract of the host represents a
reservoir from which this yeast can reinfect the vagina in
cases of recurrent vaginitis (Miles et al., 1977).
Enhanced susceptibility to adherence does not appear to be
a factor responsible for recurrent vulvovaginal candidiasis
patients who lack recognized predisposing factors, based on
comparison to normal healthy volunteers (Trumbore and
Sobel, 1986).

Other less common forms of candidiasis include
endocarditis, pulmonary candidiasis, mucocutaneous
candidiasis and esophageal candidiasis. The most common

predisposing factors include generalized debility and other

11

conditions in which the host is immunocompromised in some
way (Rippon, 1982). For example, esophageal candidiasis is
one of the most common initial presenting conditions in
patients with acquired immunodeficiency syndrome (AIDS)
(Conant, 1987). Esophageal or oral candidiasis are
conditions often present in patients with terminal
neoplastic disease, or those who are being treated with
antibacterial agents, corticosteroids or antimetabolic
drugs (Braunswald et al., 1987). Prompt, aggressive
treatment is a ‘necessity to 'not only’ prevent systemic
dissemination of the candidiasis, but also to prevent
further deterioration of the patients immune status

(Braunswald et al., 1987).

W
Host defense mechanisms against Q. m involve

both cell mediated and humoral immunity as well as non-
specific immune mechanisms (Rogers and Balish, 1980).
Antibodies to Q. amicana are found in most individuals
without a known prior history of Qandida infection (Gough,
.1984). It is postulated that the persistence of (Q.
AIIUJEUEE in the gastrointestinal tract generates an
antibody response in most individuals (Lehnmann and Reiss,
1980) , even though other microorgansism that also reside
in the gastrointestinal tract in higher numbers than Q.

Ma do not invoke a similar immune response (Berg and

12

Savage, 1972). The density of Q. 'ca 5 in the
gastrointestinal tract is affected by the intestinal flora
and. diet (Rippon, 1982). Certain. piliated strains of
bacteria such as Klanaialha can enhance the attachment of
Q. AIM to epithelial cells (Centeno et al., 1983),
while increased Qandida adherence in the presence of
Eadnazidnia 2211 is mediated by the presence of fimbriae on
the bacterial cells (Makrides and MacFarlane, 1983).
Specific anti-candida antibodies are present in non-
colonized patients, infected carriers of Q. dlhiQdES
without evidence of candidiasis, subjects with acute
candidiasis and subjects with chronic candidiasis (Epstein
et al., 1982). The titers of these antibodies reflect the
degree of antigenic stimulation, and are significantly
higher in candidiasis than in controls or colonized
carriers (Epstein et al., 1982) . Other researchers have
found that titers from patients with Qandida infections are
often within the same range as those individuals without a
Qandida infection (Gough et al., 1984). In in m
studies, Scheld et al., 1983, found that humoral antibody
protected against Q. alnidana, endocarditis through
inhibition of adhesion. In patients with mucocutaneous
candidiasis with a primary macrophage dysfunction leading
to impairment of specific cellular immune responsiveness,

carbohydrate antigens, essentially mannan, persisted in the

13

patients serum and inhibited the specific Qandjda-induced
proliferation of lymphocytes and antibody (Fischer et al.,
1982).

Bodily secretions have also been found to contain
anti-candida antibody. Vaginal secretions contain IgE and
IgA antibody classes to Q. albicans (Gough, 1984), and lack
of IgA in cervicovaginal secretions has been found to be
related to the increased adherence capacity of Q. alnidana
to vaginal epithelial cells in mm (Romero-Piffiguer et
al., 1985). In a study by Vudhichamnong et al., 1982,
secretory IgA isolated from human breast milk inhibited the
adherence of Q. alnidast to human oral epithelial cells by
blocking surface sites on Q. m, depending on the
content and concentration of specific candidal antibody.
This activity however could not be attributed solely to
specific agglutination properties of the surface IgA.
Conversely, nonspecificically bound surface IgA enhanced
adherence of the yeast and impaired the immune disposal of
Q. alLidana. In another study, a significant inverse
correlation was found between salivary IgA anti-candida
antibody and the adherence of Q. alnigang to buccal
epithelial cells, suggesting that IgA antibody can inhibit
adherence of Qandida to oral mucosa (Epstein et al., 1982).

Polymorphonucleocytes (PMN'S) (Diamond, 1981) and

macrophages (Lehrer and Fleishman, 1982) destroy Q.

14

alnidana as part of their nonspecific immune function
against microinvasion of the host. In a study by Bistoni
et al., 1986, pretreatment of mice with a strain of Q.
alnidang incapable of yeast-mycelial conversion conferred
protection against subsequent challenge with pathogenic Q.
alhidana strains. This was accompanied by an increase in
PMN's in peripheral blood and activation of splenic cells
with highly candidacidal reactivity. Adoptive transfer of
plastic adherent cells from pretreated mice to
histocompatible recipients conferred protection against
subsequent pathogenic strain challenge. In another study,
PMN's from. patients suffering from nodular lepromatous
leprosy who were deficient in cellular immunity and from
healthy controls had the same capacity to phagocytize and
destroy Q, alnidana, indicating that other defense
mechanisms are operational in dealing with infectious
agents in patients with depressed cellular immunity (Hobers
et al., 1986). Baccarini et al., 1983, also found that
phagocytic killing involved mmltiple mechanisms such that
candidacidal activity was found early in inflamation and
was due to PMN's, adherent spleen cell populations, and
macrophages to a limited extent. In another study, antigen
specific activation of human T-cells using a Q. almdang
polysaccharide extract required both antigenic

presentation and interleukin-I production (Lombardi et al.,

15

1984). In the absence of functional T-cells and viable
bacterial flora, athymic and heterozygous littermateOmice
(adult and neonatal BALB-C) colonized with pure cultures of
Q. alnidan§_ manifested resistance to extensive
mucocutaneous and systemic candidiasis (Balish et al.,
1984). In a clinical study by Gardner et al., 1984,
phagocytic cell function in the elderly did not decline at
the same rate as the specific antibody immune response,
indicating that the increased incidence of candidiasis

with increasing age is associated with multiple host

defense factors.

YIBQLEEQE
Candida alhidana and other Candida Sp- are 5-7 mm in

size and grow as creamy smooth colonies although older Q.
alnidang colonies can be wrinkled and folded in appearance
(Beneke and Rogers, 1980). Qandida species fonm mycelium
and pseudomycelium under specific environmental conditions
and when grown on specific media (Beneke and Rogers, 1980).
Pseudomycelium is composed of elongated, undetached spores
that may have clusters of blastoconidia at constrictions
(Beneke and Rogers, 1980). The presence of mycelial forms
in tissue connotes that colonization of the host has
occurred (Rippon, 1982).

The mycelial form is considered to be the pathogenic

or parasitic stage (Rippon, 1982). In one study epithelial

16

membranes were penetrated by hyphae but not blastoconidia
(Hewlett and Squier, 1980), although in tissue both forms
are found (Rippon, 1982). Studies of oral candidiasis show
that the filamentous form of Q. alnidana is necessary to
penetrate epithelial cells (Martin et al., 1984; Montes and
Wilborn, 1968). In a study using a rabbit model of renal
candidiasis, the pseudohyphal form of Q. alnidana was
necessary to cause a fatal infection (Whittle and Gresham,
1960), while in another study of renal candidiasis caused
by Q. albidana, growth of a germ tube into renal tubules
provided an advantage for amplification of the organism
(Barnes et al., 1983). Some investigators have found that
the ability to become filamentous allows Q. anginang to
escape destruction in professional phagocytes (Arai et al.,
1977) . Others have found that the formation of germ tubes
by Q. amidana is a mechanism by which this organism
escapes from phagocytic cells by conferring greater
resistance to destruction by neutrophils and macrophages,
as compared to the blastoconidial form (Culter and Poor,
1981) . Other researchers believe that both yeast and
mycelial forms of Q. alpidang can adhere, invade and
proliferate in an infected host (Shepherd, 1985) and that
the capacity of Q. alnigana to produce hyphae appears to be
an important but nonessential virulence factor in the

pathogenesis of candidal vaginitis (Sobel et al., 1984).

17

In addition, a recent study showed that Q. alnidana and Q.
tzdnidalia were capable of initiating tissue invasion
before germ tubes had the opportunity to form and
participate in the invasive process (Klotz et al., 1983).

Qandida sp. and IQIBlgpdifi glannana, as well as other
pathogenic yeasts, can be cultured from clinical specimens
using Sabourauds Dextrose Agar as an isolation. medium
(Beneke and Rogers, 1980). Identification of these yeasts
involves demonstration of sugar assimilation patterns or
secretion of enzymes specific for the organism in question
(Beneke and Rogers, 1980; Sherris et al., 1984).
Commercial yeast identification systems such as "API" or
”Yeast Ident" are used in clinical microbiology
laboratories for the identification of these organisms. In
addition, Q. aldigang is the only organism which forms
germ tubes when incubated in rich media or serum for one
hour at 37°C (Beneke and Rogers, 1980) , and thus can be
identified with this technique.

The virulence of Qandida sp. in causing disease is
determined by' many factors including the immunological
state of the host and characteristics of the organisms,
including the ability to produce extracellular enzymes
which aid in attachment and penetration of epithelial
surfaces (Rippon, 1982) . Although Q. alnidans produces

several proteinases (Ruchel, 1984), there is evidence that

18

only one of these enzymes is a virulence factor (MacDonald
and Odds, 1983) . Q. alnidans produces phospholipases
(Banno et al., 1985; Sammaranayake et al., 1984) which aid
in the penetration of hyphae into epithelial cells (Howlett
and Squier, 1980). Only Q. alnidana produces phospholipase
activity while Q, LI2219311§. I. glahrata. and Q.
W do not (Ruchel,1984). Banno et al., 1985,
demonstrated that the mycelial form of Q. alnidang produces
twice as much phospholipase activity as the yeast form,
supporting the concept that the mycelial form of Q.
albicans is the most pathogenic form. In one study
antibody to proteinase was detected in serum and in
infected tissue with indirect immunofluorescence (MacDonald
and Odds, 1980). In a study by Ghanndum and Elteen, 1986,
all Q. alnidang isolates were found to secrete an inducible
proteinase and Q. amdang isolates which adhered most
strongly to buccal epithelial cells had the highest
relative proteinase activity and ‘were most pathogenic.
Other researchers have suggested that carbohydrases may
also play a role in pathogenesis (Elinov, 1984), and that
keratolytic proteinase activity in Q, alnidana is
important in the pathogenicity of the organism in 2.13.9

(Hattori et al., 1984).

19

W

The cell wall of fungi is very important in protecting
the organism from the environment and from host defense
mechanisms (Rippon, 1982) . Fungi can be subdivided into
two categories according to cell wall composition,
including chitin-glucan and chitin-mannan-glucan components
(SanBlas, 1982). Other chemical components such as protein
are present as well. Qandida species are included in the
chitin-mannan—glucan group along with other members of the
Cryptococcaceae and the Saccharomycetaceae (SanBlas, 1982).
Five to eight layers of the cell wall of Q. alnidang have
been identified depending on growth conditions or
cytochemical techniques utilized (Poulain et al, 1978).
Chitin has been identified as the compound composing the
bulk of the most electron transparent layer in transmission
electron microscopy studies of germ tube formation (Cassone
et al., 1973). The mycelial form of Q. albidans has three
times as much chitin and only one third as much protein as
the blastoconidial form, which may explain the greater
resistance of the mycelial form to host defense mechanisms
that allows for the persistence of the mycelial form in
vivo (Chattaway et al., 1968).

The localization of mannan in the cell wall of Q.
alnidana has been investigated using a variety of

techniques including lectins such as concanavalineA,

20

antibodies to mannan, and biochemical and histochemical
methods. Mannan is a highly branched sugar molecule with
short chains of a-1,2 linked D-mannopyranosyl residues
joined by a-1,6 linkages (Bishop et al., 1960).
Histochemically, the localization of mannan in the outer
cell wall of Q. amidana has been confirmed by Djaczenko
and Cassone, 1971; Poulain et al., 1978; and Evron and
Drewe, 1984. Cassone et al., 1978, found an outer capsule-
like component of spikey fibrillar protrusions of mannan, a
homopolymer of mannose. Hannah-protein complexes span the
entire cell wall of Q. alnidans and reach into the inner
matrix, a bulky, rigid framework. of glucan and chitin
(Cassone et al., 1978; Chattaway et al., 1976). Other
studies have shown that during the regeneration of
protoplasts of Q. amdand, radioactively labelled mannan
is not associated with the protoplast indicating that
mannan is not a structural polysaccharide of the cell wall
as chitin and glucan are (Elorza et al., 1983).

The outer mannan layer is apparently easily shed
(Montes and Wilborn, 1968; Poulain et al., 1978). Old
cultures of Q. alnidang lose their outer cell wall layers
which contain mannan (Poulain et al., 1978). Diedrich et
al., 1984, and Diamond et al., 1980, isolated a
glycoprotein that contained mannose in culture filtrates.

In a study by Brawner and Cutler, 1986, two different cell

21

surface carbohydrate determinates both containing mannose
and glucose were not expressed on very young cells and
disappeared from the cell surface of most Q. alnidang
strains with age. Poulain et al., 1978, postulated that
this loss of outer cell wall mannan in older cells could
account for the mannan antigenemia seen in patients with
invasive and disseminated candidiasis (Weiner and Yount,
1976) and the suppression of the immune response
experienced by these patients (Weiner and Coats-Stephens,
1979). In addition, antibody to mannan can be found in the
serum of patients with candidiasis, in experimental
infections in animals (Weiner and Coats-Stephens, 1979) and
in normal uninfected individuals (Lehnman and Reiss,
1980) . Circulating antigen-antibody complexes have been
detected in the serum of patients with candidiasis (Burges
et a1. , 1983) and immune complexes can be generated by

adding mannan to human serum (Reiss et al., 1981).

W

Adherence of microorganisms to biological surfaces has
been recognized as the initial step in colonization and
tissue invasion of mammalian membranes (Gibbons and
VanHonte, 1975; Reed and Williams, 1978). It is also a
mechanism by which sloughing of the organisms is prevented
during the secretion of bodily fluids, food movement, or

defecation (King et al., 1980). Specific mechanisms of

22

adherence between organisms and interface exist which
‘utilize complementary molecular structures (Marshall,
1976) . The most common mechanism of adherence based on
this principle involves lectin-carbohydrate interaction
(Dazzo, 1980; Sharon and L15, 1972), while other mechanisms
such as enzymatic interaction are recognized (Gibbons,
1977) .

Bacterial attachment to epithelial membranes rarely
occurs Zby direct contact. between ‘the bacteria and the
epithelial cell membrane (Fletcher and Floodgate, 1973) .
These organisms utilize components of their capsules,
fimbriae, or pili to complete the linkage to the host cell
(Fletcher and Floodgate, 1973). For example, Eddnanidnia

lei (Johnson et al., 1979; Ofek and Beachey, 1978; Ofek et

al., 1977), Walla nnaumdniae (Fader et al., 1979),
Raanddndnaa aanngindaa (Ramphal et al., 1980; Woods et al.,
1980). Salmonella txnhimian (Berkeley et a1-. 1981).

M (Silverblatt, 1974), and Ma (Duguid et al.,
1976) are type-1 piliated strains of bacteria which attach

to epithelial cells via surface pili or fimbriae. They
adhere in greater numbers to epithelial cells than non-
piliated organisms, and utilize a mannose specific lectin
that recognizes mannose moieties on the surface of
epithelial cells (Ofek and Beachey, 1978; Ofek et al.,

1977). Non-type-l piliated strains of bacteria possess

23

capsular K antigens which resemble pili morphologically but
are protein in nature which mediate adherence to epithelial
cell surfaces (Savage, 1980). K-88 (Jones and Rutter,
1972: Berkeley et al., 1981) and K-99 (Moon et al., 1977)
antigens mediate the adherence of enterotoxigenic
W 9211 to neonatal swine small intestinal
epithelial cells. The epithelial cell receptors to these
antigens are terminal a-D-galactosyl residues of membrane
glycoproteins (Gibbons and VanHonte, 1975; Gibbons, 1977).

Other mediators of adherence in bacteria include mannose-

resistant hemagglutinin for strains of §a1_am_ona1_1a £22111
(Berkeley, et al.,1981), pili for naiadania sp.(Gubish et

al., 1979), lipoteichoic acid for alpha §£I§E§2§22£i
(Bartlett and Duncan, 1978; Ofek et al., 1975), and
extracellular polymers attached to the cell surface such as

with .5:::2§2222225..md§dn§ (Kelstrup (and Funder-Nielson,
1974) .

ADHEBEEQE_QE_QANDIDA_§EE§IE§
Adherence of Qandida sp. to biological surfaces is

important in establishing colonization and infection in the
host. It has been shown that Q. alnidand adheres to buccal
cells (Kimura and Pearsall, 1978,1980; Sandin et al.,
1982), fibrin platelet matrixes formed in_yit:d (Maisch and
Calderone, 1980, 1981), acrylic surfaces (McCourtie and

Douglas, 1981), PMN's (Diamond et al., 1978), and other

24

reticuloendothelial system cells (Warr, 1980; Stahl et al.,
1978) . Q. albidand has been found to adhere in greater
numbers than Q. M, Q. Mia, and other
Qandida sp. to vaginal cells (King et al., 1980), buccal
cells (Kimura and Pearsall, 1980; King et al., 1980;
Rotresen et al., 1986) and to fibrin platelet matrixes
formed in 21:19 (Rotresen et al., 1985). Qandida albidana
also adheres more strongly to epithelial cells than fungal
cells of other Qandida sp. (Macura 1985).

It is generally agreed that there is great variation
in the number of receptor sites for Q. albidans on
epithelial cells collected from different individuals (King
Etwavl” 1980; Sandin et al., 1987), and that day to day
variations in adherence exist in swabbings fromwamsingle
subject (King et al., 1980). Researchers have hypothesized
that indigenous flora could suppress adherence of Q.
alnidang by competing with it for receptor sites on
epithelial cells, modifying these sites to hamper candidal
adherence, or enzymatically altering the yeast surface
(Liljemark and Gibbons, 1973). For example, the
observation that females are more prone to suffer candidal
vaginitis during and after menses (Mead, 1974) when
lactobacilli are less prevalent in the vagina and pH is
increased (Saigh et al., 1978) correlates with the findings
that Q. alnidana adheres better to vaginal cells at a pH 6

25

rather than pH 3-4 in in yltzd experiments (Sobel et al.,
1981) . Other researchers have found increased candidal
adherence to small intestine cells due to the presence of
W gall which is mediated by the presence of
fimbriae on the bacterial cells and mannose-like receptors
on both the surface of Q. alnigana and epithelial cells
(Makrides and MacFarlane, 1983). Centeno et al., 1983
found that Klanalalla could also enhance the attachment of
of Q. alnldana to intestinal epithelial cells.

Nonspecific host factors which affect candidal
adherence to biological surfaces include local defense
mechanisms and environmental conditions. Oral factors such
as the epithelial barrier, salivary flow, microbial
interactions, antimicrobial constituents of saliva,
lysozyme, lactoferrin and lactoperoxidase systems, iron
levels and salivary glycoproteins have been found to affect
adherence (Epstein et al., 1984). Tear proteins including
albumin, lactoferrin, lysozyme, and fibronectin enhance
yeast adherence to both hard and soft contact lenses
(Butrus and Klotz, 1986) . Samaranayake and MacFarlane,
1982, found that exogenous sugar sources affected oral and
vaginal carriage of Q. albldand by modifying adherence
properties.

The study of the adherence of Q. alnldang to

biological surfaces has included both viable and formalized

"L-s d-..-

26

cells infdifferent growth stages. Many studies have shown
that [the germinated form of Q. alnldana adheres to
epithelial cells in greater numbers than non-germinated
formsqlléimura.-andu Pearsall, 1978, 1980; King et al., #1980“;
Sandin et al., 1982; Rotresen et al., 1986), although both
blastoconidial and mycelial forms of Q. alnldana can
adhere, invade, and proliferate in an infected host
(Shepherd, 1985) . Another study showed that the capacity
of Q. alnidana to produce hyphae appeared to be an
important but nonessential virulence factor in the
pathogenesis of candidal vaginitis (Sobel et al., 1984).
Preincubation of Q. albidans isolates in tissue-culture
medium M-199 prior to adherence favors germ tube formation
(Kimura and Pearsall, 1978, 1980; Sandin et al., 1982).
Once germinated, the cells can be killed in formalin with
no significant decrease in adherence, while treatment
before germination decreases adherence (Kimura and
Pearsall, 1978; Sandin et al., 1982). In a study of
attachment to endothelial cells, viable or killed Qandlg
organisms were enveloped by membrane processes from
endothelial cell surfaces and were incorporated into the
endothelial cells in phagosomes (Rotresen et al., 1985) .
Researchers have postulated that changes in the cell wall
of Q. alnldana as it germinates could be responsible for

the increased adherence seen (Kimura and Pearsall, 1978;

27

Sobel et al., 1981). In addition, the stationary phase of

growth of Q. alnidang has shown greater adherence values

than cells at the logarhythmic stage (King et al., 1980).
Qandlda_ albidand, produces several extracellular

enzymes which aid in adherence to epithelial cells and that

#4 7“."

contribute to the virulence of this organism. In a study
by “Ghanndum and Elteen, 1986, Q. alnidana isolates,
especially strain type-C, adhered most strongly to buccal
epithelial cells, had the highest proteinase activity and
were most pathogenic. Qandjda alblgang isolates secreted
an inducible proteinase and showed a tendency toward
greater adherence ability. In another study, Q. alnidana
isolates which adhered most strongly to buccal epithelial
cells were most pathogenic in mice and had the highest
phospholipase activity (Barrett et al., 1985).

Adherence of Q. alnidana to polymeric surfaces has
been studied and Minagi et al., 1985, found that the closer
the surface free energy of the substrate surface and the
microorganism, the higher the probability of adherence.
Forces responsible for adherence of Q. alnldana and other
Qandida sp. to inert polymeric surfaces are London-Van der
Waal forces (hydrophobic forces) and electrostatic forces,

and in this study the hydrophobicitynmofmw£h§1m¥§asts.

correlated with the tendency of yeast to adhere to

polystyrene (Klotz et al., 1985). Another study suggests

28

that several factors are involved in the adhesion of Q.

ans to plastic, and indicates that surface
hydrophobicity is of minor importance in direct adhesion to
epithelial cells but that it may contribute to indirect
attachent to epithelial cells by promoting yeast coadhesion
(Kennedy et al., 1988).

As stated previously, the mycelial form of Q. albicans
is postulated to be the pathogenic stage (Kimura and
Pearsall, 1980), although other studies have shown that all
stages of the organism can adhere and invade to cause
infection in the host (Shepherd, 1985). In scanning
electron microscopy studies, adherence of Q. algidang to
mouse vagina showed that six hours after infection short
projections of the yeast were attaching to mucosal cell
surfaces, and that later mycelium grew from the yeast
(Campisi et al., 1986). In another study Barnes et al.,
1983, found that attachment of Q. alnldana to endothelium
within the capsule of the renal cortex was a key event in
the disease process, and that growth of a germ tube into
the renal tubules provided an advantage for amplification
of Q. alnldang. In another study, Klotz et al., 1983,
found that adherence and penetration of epithelial cells in
the initiation of tissue invasion occurred before germ
tubes had the opportunity to form and take part in the

invasive process.

29

Characterization of the adhesin which mediates
adherence of Q. alnlgana to biological surfaces has been
studied extensively. Kimura and Pearsall, 1978, found that
enhanced adherence of Q. alnldans after incubation in
saliva was related to changes in the fungus itself. In a
study by Sobel et al., 1981, adherence of Q. albldans was
enhanced by a surface component of the germinated yeast,
postulated to be a glycoprotein. In scanning electron
microscopy studies yeast were associated with a mucus layer
on epithelial surfaces throughout the gastrointestinal
tract (Pope and Cole, 1981) . Another study» showed that

attachment of Q. M to buccal epithelial cells

\ 9....”er v

I [appeared to involve spatial rearrangement of their cell
V. wall surface. Adhering yeast developed a fibrogranular
surface layer which (was detected by using specific
carbohydrate staining techniques and concanavalin-A binding
that appeared to mediate attachment of yeast to epithelial
cells by a mannose-mediated receptor (Tronchin et a1. ,
1984). Cassone et a1, 1978, found that mannan is the major
constituent of the fibrillar-floccular layer of the cell
wall of Q. aldigans using con-A.
A variety of methods have been used to study the
inhibition of adherence of Q. alnidana to different
epithelial surfaces. . Pretreatment of Q. alnidand with

trypsin, chymotrypsin or proteinase decreased adherence

30

significantly (King et al., 1980; Maisch and Calderone,
1980; Silverblatt, 1974). Other researchers using an
alkali treatment method determined that the extract was
predominantly mannose (Maisch and Calderone, 1981) . Loss
of adherence after treatment of yeast cells with
a-mannosidase or papain suggests that the cell wall
mannoprotein is an essential component of the Q. alnldana
adhesin (Lee and King, 1983). In a study by Sandin and
Rogers, 1982, con-A pretreatment of the yeast inhibited
adherence, while preincubation of the lectin with d-mannose
restored adherence during the adherence assay. In
addition, addition of a-D-methylmannopyranoside in the
incubation medium during the adherence assay between yeast
and buccal epithelial cells inhibited adherence (Sandin et
al., 1982). In competitive inhibition studies using crude
and purified cell wall products, blocking antibody,
lectins, and controlled degradation of cell surface of Q.
alnidand. mannans and mannoproteins appeared to be
important constituents of the adhesin mediating adherence
(Rotresen et al., 1986).

Chitin has also been found to inhibit adherence of Q.
alnldana via N-acetylglucosamine to human vaginal
epithelial cells, leading the authors to conclude that
amino groups of sugars are responsible for inhibition of

adherence of Q. alnidana to epithelial cells (Segal et al.,

31

1982). In. another study, inhibition of adherence ‘was
produced by aminosugars including mannosamine, glucosamine
and galactosamine (Collins-Lech et al., 1984). In
addition, in experimental murine vaginitis, pretreatment of
animals with chitin derivatives blocked the attachment of
yeast to vaginal mucosal surfaces and led to the
prevention of vaginal infection (Lehrer et al., 1983).
Scanning electron microscopy studies have also shown
non-uniform distribution of adhering microorganisms with
diminished adherence in areas of active mitosis and
proliferation, and increased adherence to mature flat cells
often in the process of desquamation (Sobel et al., 1982).
Other studies have shown increased Q. alnldana adherence in
situations where there is an increase in the number of
intermediate epithelial cells (Segal et al., 1984), such as
during pregnancy, and the first and fourth weeks of the
menstrual cycle (Robbins et al., 1982), indicating that
epithelial cell characteristics also affect adherence.
Elucidation of the adherence mechanisms utilized by Q.
alnldana and other Qandida sp. in causing disease is
important in research and development of new treatment
methods. Drugs used in the treatment of candidiasis or

experimentally _ have been used to, study ...adherence of Q.

alnldang _m.-Amphotericin B is a ,fungal. cell membrane. ,

synthesis inhibitor' which. is used_ in 'the rtreatment of

32

candidiasis (Katzung, 1985). When used in adherence
inhibition studies, amphotericin B inhibited adherence
after 3-72 hours of exposure time at MIC or sub-MIC values.
In comparison, 5-fluorocytosine, nystatin, miconazole and
ketoconazole which all are drugs used in the treatment of
candidiasis (Katzung, 1985), interfered with adherence only
after 72 hours and at levels greater than MIC (Brenciaglia
et al., 1986). Other studies with amphotericin-B showed a
protective effect in mice receiving a single
intraperitoneal injection which was dependent on dose,
time of drug administration and size of yeast innoculum.
These mice also showed resistance to subsequent challenge
with Q. alnlgang. Effector cells and macrophages from
animals exposed to amphotericin-B show strong candidacidal
reactivity (Bistoni et al., 1985).

Other drugs have also been used to study candidal
adherence. For example, chlorhexidine is another
antifungal agent which in one study decreased adherence by
19-86% in. treatment. of saliva. or serum. coated acrylic
persisting up to 19 days after exposure. Adherence was
also significantly decreased by pretreatment with
chlorhexidine under conditions which. enhanced adherence
including stationary phase of growth, high galactose, and
high sucrose (McCourtie et al., 1986). Noxythiolin,

another antifungal agent, decreased adherence in both

33

exponential and stationary growth phases of both the
blastoconidial and pseudohyphal forms, as well as when only
the epithelial cells were treated with the drug ( Gorman et
al., 1986). In a clinical study of the use of nystatin,
patients receiving two weeks of therapy had marked
improvement in the signs and symptoms of candidiasis as
well as a significant reduction in the number of candidal
organisms present although the condition recurred rapidly

following cessation of treatment (Epstein et al., 1981).

IMATERIALS AND METHODS

BHQQAL_HHQQ§AL_QELL§
Buccal cells were collected by gently rubbing the

inside cheek area of six healthy adult volunteers with
sterile swabs and swirling the swabs in phosphate buffered
saline (PBS), pH 7.2 (Sandin et al., 1982). Donors had no
signs or symptoms of oral thrush, were culture-negative for
yeast (pharyngeal specimen) and were not taking antibac-
terial or antifungal agents at the time. The individual
specimens were washed three times in PBS and then
separately suspended to concentrations of 2 x 105 viable
cells per ml of PBS, as determined by methylene blue

staining and hemocytometer count.

QBEANIfiMfi
Organisms used included clinical Qandlda alnm

isolates MSU-l, 1840, UCLA, DA-05900, CBC-1134, 10-16-35F,
a. naranaildsia. Q. traniaalis. c. Wis. Q-
atellatdidea. Micaela dLaIzIaLa-l. and SI:- dlahrata-B (See
Appendix A-I for clinical source of isolates). Each
organism was grown on Sabauraud's Dextrose Agar (BBL,
Cockeysville, MD, USA) slants at 37°C for 24-48 hours. A
loop of cells was transferred to 100 ml of Trypticase Soy
Broth (BBL Microbiology Systems, Cockeysville, MD) plus 4%
glucose and incubated at 37°C on a rotary shaker (180 rpm)

for approximately 15 hours to develop the stationary phase

34

35

of growth. The cells were then washed three times with PBS.
Qandida alnidana isolates were resuspended in tissue-
culture medium M199 (Gibco Laboratories, Santa Clara, CA)
adjusted to pH of 7.2. These suspensions were incubated
for one hour at 37°C for the developement of germ tubes
(Sandin et al., 1982) . Yeast with and without germ tubes
were counted to determine the percentage of cells which had
germinated. The organisms were then washed three times in
PBS. For all organisms, approximately seven eighths of the
yeast (germinated for Qandida albidand isolates) were
retained for viable cell studies and for cell treatment
methods to be used in adherence studies. The remaining
cells were resuspended in 0.5% formaldehyde in PBS for 30
min. at 4°C to kill the cells. After removal of the
formaldehyde the yeast were washed three times in PBS.
Viable and formalized preparations of the organisms were
resuspended at 106 viable cells per ml of PBS, as
determined by methylene blue staining and hemocytometer
count. See Appendix A-II for diagram of organism

preparation.

W

In the adherence assay 0.2 ml samples of buccal and
yeast cells were pipetted into 12 x 75 mm test tubes and
incubated on a shaker at 180 rpm for 1 hr. at 37°C. Three

tubes were used for each control (buccal cells only in

36

tube) and for each experimental test. Polycarbonate
filters (12 um pore size; Nucleopore Corp., Pleasanton,
CA) were used for collection of the adherence mixture from
each tube and. washed. with 100 :ml PBS ‘under continual
agitation. These filters were used because the pore size
allows only non-adhering yeast to go through, while buccal
cells with adhering yeast remain. The filters were stained
with Gram's Crystal Violet and the number of yeasts
adhering to 100 buccal cells was determined by light
microscopy at 430x. Double-blind conditions were used in
all studies (Sandin et al., 1982). (See Appendix A-III for

diagram of adherence procedure.)

IBEAIMEKI__M§T§QQ§ (See Appendix A-IV for diagram of

treatment method protocols.)

QQHQAHA!AL1H;A: Concanavalin-A (Con-A) was used at a
concentration of 10 ug/ml of PBS containing added cations
(0.002 M Magnesium, Calcium, and Manganese salts), (Sandin
et al., 1982). Con-A was obtained from ICN Pharma-
ceuticals, Cleveland, OH. A 5.0 ml sample of the
germinated. yeast (Qandida_.albidana isolates) or ‘viable
yeast were pelleted and suspended in 15.0 ml of Con-A
solution and incubated at room temperature on a shaker for
45 minutes. The cells were washed and resuspended in PBS

to 5 ml and mixed with buccal cells for the adherence

37

assay. All steps were carried out under conditions of

continual agitation to prevent agglutination.

W: 200 mg Of a-D-methylmanno-
pyranoside (A-DM) was dissolved in 0.5 ml PBS and added to

adherence test tubes that already contained a total of 0.4
ml of the standardized yeast and buccal cell suspensions,
or to the control tubes. The contents of each tube were
mixed on a Vortex mixer and were immediately set on a

shaker at 180 rpm to proceed with the adherence test.

W: A 5.0 ml sample of the germinated or viable
yeast was pelleted and resuspended in 15.0 ml of a 0.1 M,
pH 7.0) solution of citric acid in PBS, autoclaved for 1
hr. at 120°C, washed three times in PBS, and resuspended to
5.0 ml in PBS (Tronchin et al., 1984). These cells were

then mixed with buccal cells for the adherence assay.

W: A 5-0 ml sample 0f the

germinated or viable yeast preparation was pelleted and
resuspended in 15.0 ml of a 40 ug/ml solution of
filamentous hemagglutinin (FHA) (courtesy of Dr. Larry
Winberry, Michigan Department of Public Health, Biologic
Products Division) and incubated for 45 min. on a shaker at
room temperature. The cells were washed three times in PBS
and resuspended to 5.0 ml in PBS. They were then mixed

with the buccal cells for the adherence assay. All steps

38

were carried out under conditions of continual agitation to

prevent agglutination.

SIAIIEIIQS

The results were analyzed using a two way analysis of
variance where two factors are considered simultaneously
and the two sources of variance are of equal rank.
(Robert, et al., 1981). Mean and standard deviation

calculations were also completed where appropriate.

RESULTS

Qandida alnidans isolates were subjected to conditions
which permitted germ tube formation and used in the
adherence assay. Adherence represents the average number
of yeast adhering to 100 buccal epithelial cells in six
different human buccal epithelial cell donor samples, in
triplicate. Q. allaic_an§ isolate MSU-l (99% germ tubes)
had an average of approximately 69 yeast cells adhering to
100 buccal cells in each donor sample, while isolate 10-16-
35F (80% germ tubes) had an average of 64 adhering yeast
per 100 bucccal cells. Isolate ONC-1134 (71% germ tubes)
had approximately 60 adhering yeast, DA-05900 had an
average of 57 yeast adhering, UCLA had approximately 47
yeast adhering, and 1840 had an average of 40 adhering
yeast per 100 buccal cells (Figure 1). As germ tube
formation capability increased for Q. albidana isolates,
adherence increased. The Q. aldidana strains used were
clinical isolates (Appendix A-I).

Adherence of Qandida spp. and W glam
isolates to human buccal epithelial cells was examined
using germinated Q. alnldana and non-germinated alternate
Qandlda spp. and I. glanLaQa isolates. (The number in
parenthesis after each isolate represents the range of the
average number of yeast adhering to one hundred buccal
epithelial cells in six different human buccal epithelial
cell samples tested.) Q. alnjgand (69) adhered to a

39

FIGURE 1 .

40

Comparison of adherence of Qandlda
albicans isolates of differing germ
tube formation capability to human
buccal epithelial cells. (Percent
adherence for each organism determined
by averaging percent adherence of
formalized and viable yeast to
epithelial cells.) Organisms

include:

a. 1840, 5% germ tubes

b. UCLA, 52% germ tubes

c. DA-05900, 65% germ tubes
d. ONC-ll34, 71% germ tubes
e. 10-16-35F, 80% germ tubes
f. MSU-I, 99% germ tubes

WOZMWMICUP

75-1

70-

65—

60-

55-

50-

45-1

40—-

35-

no

 

 

 

 

41

In

 

 

 

 

JD.-

 

 

 

]m

 

 

1H.

 

 

 

 

30

PERCENT GERM TUBES

70-

 

80

90

100

42

greater extent than I. 9].dede isolates (30-33) or other
Qandlda spp. (15-25) to human buccal epithelial cells, with
I. glannata isolates (30-33) adhering greater than (in
order of decreasing adherence) Q. Mia (25), Q.

e o 'c (22), Q. Mia (19): or Q.
W (15) (Figure 2). Viable organisms
(germinated Q. albicans) were used in the adherence assay.
Organisms used in this study were clinical isolates (see
Appendix A-I) and were chosen based on frequency of
isolation in patient populations with yeast colonization
and infection.

Figures 3-14 demonstrate the effect of various
treatment methods on adherence of six Q. alnidana isolates,
two I. diaarata strains. Q-__s_tellat_o_idea. S;- naransiloaia.
Q. W, and Q. psandgtmidalia to six different
human buccal epithelial cell samples. Treatment methods
included formalization (FY) which killed the organisms,
treatment with citric acid (CY) which decreased cell wall
mannan, filamentous hemagglutinin treatment (FHA) which
bound cell wall mannan and functioned as a mannan
inhibitor, concanavalin-A treatment (CON-A) which also
bound cell wall mannan and also functioned as a mannan
inhibitor, and treatment of the adherence mixture with
a-D-methylmanno pyranoside (A-DM) which as a complementary

sugar, inhibited mannan. Viable yeast were used for all

43

FIGURE 2. Comparison of adherence of clinical

isolates of Qandida sp. and Tagalonsis

glabrata to human buccal epithelial
cells.

rd (3 :3 Id PU Cd 5: C7 3>

80—

70-—

60-

50—4

40—.

30..

20—

10—

 

 

 

 

 

44

 

 

 

 

 

 

 

 

 

 

 

“_ndide
albicans

ISO-l

Torulgpaia
11:29.91

Torulopaio
.labrata 3

Candida
tro 1-

 

El»

 

Candida

m
“413

Candida
Italia-
toidca

dam;

8110013

FIGURE 3.

45

Comparison of the effect of different
treatment methods on adherence of
Qandida albidans isolate MSU-I

to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan-resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

46

2rn<

<Izoo

mnomfimz.fizmzH¢mmH

<mb

we

 

 

++++++

++++++

 

+
+++

 

++

 

++++

+ ++++

 

 

ll ON

 

Ailom

dammmmzom

47

FIGURE 4. Comparison of the effect of different
treatment methods on adherence of
Qandida alnidana isolate 10-16-35F
to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

48

:12

$28

mQOEZ HZNEfiE

st

s so

 

 

++++

 

+++++

 

 

 

 

Ion

lo.»

low

low

Ten

 

<DEMM§JZOW

FIGURE 5.

49

Comparison of the effect of different
treatment methods on adherence of
Qandida albldana isolate ONC-1134

to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

50

is

macaw: Hzmzhfimg

<nzou «as

e s

Mum

 

 

+ ++++++
.. :1.

 

 

++++

+ ++++

 

 

 

ION

tom

10m

too

 

tom.

<ammmmzom

FIGURE 6.

51

Comparison of the effect of different
treatment methods on adherence of
Qandlda albicans isolate DA-05900

to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

52

Since.

728

magnum: Hzmfigyfi.

$5

 

 

+ You
+ + +
++ + ..
+ ++ ++ ++ + + r
+ + + ++
+
+
+ + + too
+ 1 +
_

 

 

 

 

 

 

<Q§3MMIIIZUBJ

 

 

 

FIGURE 7.

53

Comparison of the effect of different
treatment methods on adherence of

Candida albicans isolate UCLA

to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills

yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhiition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

54

:JD<

$28

maomkmz Hzmzadmmfi

<mm

M>

wo

 

 

++++++

 

++n+++

 

 

++

++

 

+ ++++

 

++++

ll o~

II On

fil.om

lien

 

luoo

<:c:=:n1a:n:2:L>nJ

55

FIGURE 8. Comparison of the effect of different
treatment methods on adherence of
Qandida albicans isolate 1840
to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

56

ZID<

«Tees

maomfimz HZMZH<M¢H

a...

>>

a

 

 

+++++

 

d

 

++

++ +

 

 

 

.ll.o~

i. 8

Iluom

ll_oq

 

('Qtfll-IJMBJZUN

FIGURE 9.

57

Comparison of the effect of different
treatment methods on adherence of
Tozdlonsis glannata isolate #1

to six different human buccal epithelial
cell samples.* Treatment methods

include:
FY:

CY:

VY:

formalin treated yeast (kills
yeast)

yeast treated with citric
acid (decreases cell wall
mannan)

viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside

treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

58

ZIQ<

dlzoo

moomHmz Hzm29<mmfi

is

>>

yo

wk

 

 

 

:1

 

+

 

 

 

 

 

 

 

 

 

 

Ina

tom

1mm

ion

1mm

 

<Q=CMO§IIJZUIIJ

FIGURE 10.

59

Comparison of the effect of different
treatment methods on adherence of
Tagalonsis glanzata isolate #3

to six different human buccal epithelial
cell samples.* Treatment methods
include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

60

Elm?

72.8

macaw: Hzmadumm.

g

g

No

 

 

+++++

+++++

 

++

 

+++

s

+++

 

 

+++ ++

 

[mm

ION

IMN

Tom

1mm

 

toe

<D=CIBJD§BJZO£IJ

61

FIGURE 11. Comparison of the effect of different
treatment methods on adherence of
Qandida §£§lld£91d§d_t0 four different
human buccal epithelial cell samples.*
Treatment methods include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

62

ZIO<

$28

maomHmz 92m29<mmh

<mh

we

 

 

 

3+

 

 

TL

 

 

I! OH

l.ma

:.ON

 

<QE§JO€WZU§J

FIGURE 12.

63

Comparison of the effect of different
treatment methods on adherence of
Qandida Ed:d2§il§§i§.t0 four different
human buccal epithelial cell samples.*
Treatment methods include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

64

:12

<Izoo

maomsmz HzmzH<NMH

dim

w>

MM

 

 

 

 

3+

 

 

?

fr

 

 

 

II Ofi

II mg

IION

limN

 

<Q2CLIJMIIJZUBJ

FIGURE 13.

65

Comparison of the effect of different
treatment methods on adherence of
Qandida LIQEileifi to four different
human buccal epithelial cell samples.*
Treatment methods include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

66

Sin—4

. $.28

moomhm: Ragga

$5

w>

Wm

 

f;

 

 

 

++++

t:

 

 

 

++++

 

++++

_..|O~

[.2

 

<Q=CIflMIIJZUFfl

67

FIGURE 14. Comparison of the effect of different
treatment methods on adherence of
Qandida ngadddtndnidalls to four different
human buccal epithelial cell samples.*
Treatment methods include:

FY: formalin treated yeast (kills
yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan) ,

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (binds mannan
resulting in inhibition)

Con-A: concanavalin-A treated yeast

(binds mannan resulting in
inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar
mannan inhibition).

* The vertical error bars indicate the
standard deviation within individual
buccal cell samples.

68

ZIO<

<Izoo

maomhmz Hzm29<mmh

M>

>0

 

 

 

++-+

++++

 

 

d

t...

 

 

++++

IIOH

lma

 

f8

<Ommmmzom

69

treatment method preparation procedures. Qandlda alnldana
isolates were germinated prior to treatment and use in the
adherence assay, while other organisms used were in the
blastospore stage of development. Buccal cells were
collected immediately prior to use in the adherence assay,
washed, and resuspended. Buccal cell donors included three
males (ages 32-66) and three females (ages 22-24).
Adherence for Qandlda alnldana isolate MSU-I (99% germ
tubes) viable yeast (63.0-72.3) was similar to and did not
significantly differ from adherence for formalized yeast
(60.7-70.7) (Figure 3, Appendix B-I, Appendix C).
Adherence for CY (17.7-25.7), FHA (10.7-15.7), Con-A
(16.7-20.3), and A-DM (17.0-22.7) treatment methods was
significantly lower than fermalized or viable yeast
preparations. These treatment methods did not signifi-
cantly differ statistically from each other in terms of
effect on adherence, although FHA treatment did decrease
adherence to the greatest extent. (The numbers in
parenthesis for each treatment method represent the range
of the average number of adhering yeast to six different
epithelial cell donor samples. This applies to all Q.
amalgam and I. glabrata isolates.) Variations between
individuals for each treatment method were not significant
in most trials, although in some instances, differences did

exist which were significant. For instance, the adherence

70

of the formalized yeast preparation to cells from donor #3
significantly differed from adherence of the formalized
yeast preparation to donor #5 cells (Appendix C).

Adherence for Q. alnldang isolate 10-16-35F (80% germ
tubes) viable yeast (64.7-68.3) was similar to and did not
significantly differ from adherence for formalized yeast
(61.3-67.3) (Figure 4, Appendix B-I, Appendix C).
Adherence for CY (21.7-26.7), FHA (17.7-20.0), Con-A
(21.3-24.3), and A-DM (21.0-24.3) treatment methods was
significantly lower than formalized or viable yeast. These
treatment methods did not significantly differ from each
other in terms of effect on adherence, although FHA did
decrease adherence to the greatest extent. variations in
adherence between individuals for each treatment method
were usually not significant, although in some instances,
differences were noted. For example, adherence of viable
yeast to donor #5 cells significantly differed from
adherence of viable yeast to cells from donor #6 (Appendix
C).

Adherence for Q. alnidang isolate ONC-1134 (71% germ
tubes) viable yeast (53.7-64.3) was similar to and did not
significantly differ from adherence for formalized yeast
(52.0-63.3) (Figure 5, Appendix B-I, Appendix C).
Adherence for CY (18.7-26.0), FHA (14.0-19.3), Con-A

(16.3-25.0), and A-DM (18.0-25) treatment methods was

71

significantly lower than formalized or viable yeast. These
treatment methods did not significantly differ from each
other in terms of effect on adherence, although FHA did
decrease adherence to the greatest extent. variations in
adherence between individuals for each treatment method
were usually not significant, although in some instances,
differences were noted. For example, adherence of
formalized yeast to buccal cell donor #1 cells
significantly differed from adherence of formalized yeast
to cells from donor #5. Similar differences in other
treatment methods between individuals were also noted (see
Appendix C).

Adherence for Q. alnldans isolate DA-05900 (65% germ
tubes) viable yeast (59.0-63.3) was similar to and did not
significantly differ from adherence of formalized yeast
(48.7-60.3) (Figure 6, Appendix B-I, Appendix C).
Adherence for CY (27.3-38.3), FHA (20.0-30.3), Con-A
(26.3-37.3), and A-DM (26.7-36.3) treatment methods was
significantly lower than formalized or yiable yeast. These
treatment methods did not significantly differ from each
other in terms of effect on adherence, although FHA did
decrease adherence to the greatest extent. Variations in
adherence between individuals for each treatment method
were usually not significant, although in some instances,

differences were noted. For example, adherence of

72

formalized yeast to donor #2 cells significantly differed
from adherence of formalized yeast to cells from donor #5
(Appendix C).

Adherence of Q. amdana isolate UCLA (52% germ tubes)
viable yeast (44.0-53.7) was similar to and did not
significantly differ from adherence for formalized yeast
(44.7-48.0) (Figure 7, Appendix B-I, Appendix C).
Adherence for CY (19.0-23.0), FHA (15.0-17.0), Con-A
(18.7-22.3), and A-DM (20.3-22.7) treatment methods was
significantly lower than formalized or viable yeast. These
treatment methods did not significantly differ from each
other in terms of effect on adherence, although FHA did
decrease adherence to the greatest extent. Variations in
adherence between individuals for each treatment method
were not significant (Appendix C).

Adherence for Q. alnldang isolate 1840 (5% germ tubes)
viable yeast (36.3-45) was similar to and did not
significantly differ from adherence for formalized yeast
(33.0-41.0) (Figure 8, Appendix B-I, Appendix C).
Adherence for CY (6.0-13.3), FHA (5.3-9.3), Con-A (7.7-
12.0), and A-DM (8.3-12.3) treatment methods was
significantly lower than formalized or viable yeast. These
treatment methods did not significantly differ from each
other in terms of effect on adherence, although FHA did

decrease adherence to the greatest extent. Variations in

73

adherence between individuals for each treatment method
were usually not significant, although in some instances,
differences were noted. For example, adherence of viable
yeast to donor #5 cells significantly differed from
adherence of viable yeast to cells from donor #3 and #4
(Appendix C).

Adherence for I. glabrata #1 viable yeast (30.3-32.0)
was similar to and did not significantly differ from
adherence for formalized yeast (27.3-29) (Figure 9,
Appendix B-II, Appendix C). Adherence for CY (23.7-27.3),
FHA (16.7-19.7), Con-A (22.3-27.0), and A-DM (23.0-26.7)
treatment methods was significantly lower than fbrmalized
or viable yeast. These treatment methods did not signifi-
cantly differ from each. other' in ‘termis of’ effect of
adherence, although FHA. did decrease adherence to the
greatest extent. Variations in adherence between indivi-
duals for each treatment method were not statistically
significant (Appendix C).

Adherence for I. glannata isolate #3 viable yeast
(34.3-36.8) was similar to and did not significantly differ
from adherence for formalized yeast (30.0-32.0) (Figure 10,
Appendix B-II, Appendix C). Adherence for CY (26.0-28.3),
m (17.7-20.0), Con-A (25.7-27.7), and A-DM (25.0-27.0)
treatment methods was significantly lower than formalized

or viable yeast. These treatment methods did not signifi-

74

cantly differ from each other in terms of effect on
adherence, although FHA. did decrease adherence to the
greatest extent. Variations in adherence between
individuals for each treatment method were not
statistically significant (Appendix C).

Adherence for Q. fiEdlldIQifidd viable yeast (25.3-26.3)
was similar to and did not significantly differ from
adherence for formalized yeast (23.7-25.3), CY (19.7-22.0),
Con-A (20.3-21.7), and A-DM (19.7-22.3) (Figure 11,
Appendix B-III, Appendix C). Adherence of FHA treated
yeast (13.7-14.7) was lower than and significantly differed
from all other treatment methods (Appendix C). (The
numbers in parenthesis after each treatment method
represent the range of the average number of yeast adhering
to 100 buccal cells in four different donor samples. This
applies to non-Q. alnldana Qandlda spp. isolates.)
‘Variation in adherence between individuals for each
treatment method were usually not significant, although in
some instances, differences were noted. For example,
adherence of A-DM yeast to donor #1 cells significantly
differed from adherence of A-DM yeast to cells from donor
#3 (Appendix C).

Adherence for Q. nananalldala viable yeast (21.0-22.7)
was similar to and did. not significantly differ from

adherence for formalized yeast (20.3-20.7), CY (17.7-19.0),

75

Con-A (18.0-19.0), and A-DM (14.3-14.7) (Figure 12,
Appendix B-III, Appendix C). FHA treated yeast (13.0-14.0)
adhered the least, and significantly differed from all
other treatment methods. variations in adherence between
individuals for each treatment method were not
statistically significant (Appendix C).

Adherence for Q. 32122191113 viable yeast (16.3-17.3)
was similar to and did not significantly differ from
adherence for formalized yeast (15.3-15.7), CY (14.3-14.7),
Con-A (14.3-14.7), and A-DM (14.3-14.7) (Figure 13,
Appendix B-III, Appendix C). FHA treated yeast (9.7-10.3)
adhered the least, and significantly differed from all
other treatment methods. variations in adherence between
individuals for each treatment method were not
statistically significant (Appendix C).

Adherence for Q. naanddtzdpidalla viable yeast (18.7-
20.3) was similar to and did not significantly differ from
adherence for formalized yeast (17.7), CY (15.7-16.7),
Con-A (15.7-16.7), and A-DM (15.0-16.0) (Figure 14,
Appendix B-III, Appendix C). FHA treated yeast (13.0-14.3)
adhered the least, and significantly differed from all
other treatment methods. variations in adherence between
individuals for each treatment method were not

statistically significant (Appendix C).

76

Overall, there was little variation in adherence of a
particular organism preparation to an individual buccal
cell sample (tests were done in triplicate) (Figures 3-14,
Appendix B and C). Similarly, there was little variation
in adherence of a particular organism preparation to all
samples of buccal epithelial cells, although variations did
exist in some cases, especially for epithelial cell donor
#5 in most organism preparations where a difference exists.
Formalized and viable yeast preparations did not
statistically differ in adherence to buccal cells for each
organism (see Appendix C for ANOVA results). Other
treatment methods decreased adherence, with FHA treated
cells adhering the least for all organisms, in comparison
to other treatment methods. In some instances, even though
FHA did decrease adherence to the greatest extent, this
amount did not statistically differ from the decrease in
adherence seen with other treatment methods used. CY, Con-
A, and A-DM effects on adherence were not different
statistically within treatment groups for a particular
organism tested (see Appendix C for ANOVA results). For

statistical analysis of data for each organism, refer to

Appendix B (Candida albicans isolates, B I: mums
glabrata isolates, B II; other Qandida spp., B III).

Figure 15 represents a comparison of adherence after

treatment methods, explained previously, of germinated

FIGURE 15.

77

Comparison of effects of treatment
methods on adherence of.£andida_
albicans strains to human buccal
epithelial cells. Organisms
include:

- MSU-I

A 10-16-35-F
- ONC-ll34
+ DA-059OO
0 UCLA
x 1840

a Treatment methods include:

FY: formalin treated yeast
kills yeast)

CY: yeast treated with citric
acid (decreases cell wall
mannan)

VY: viable yeast

FHA: filamentous hemagglutinin

treated yeast (mannan
inhibition)

Con-A: concanavalin-A treated

yeast (mannan inhibition)

A-DM: a-D-methylmanno

pyraonside treated
adherence mixtures
(complementary sugar
mannan inhibition)

mozwwmmu»

80—

70—

60-

50—

40-

20—1

10—

78

 

 

 

VY CY FHA CON-A AD-M

TREATMENT METHODS

79

Candida albicans isolates to pooled human buccal epithelial
cells (six donors). (The numbers in parenthesis following
each treatment method represent the average number of yeast
the standard deviation adhering to 100 pooled buccal
epithelial cells.) FHA treatment of cells decreased
adherence to the greatest extent as compared to Con-A,
A-DM, or CY samples, which decreased adherence approxi—
mately equally within trials for each organism. For
example, adherence for Candida albicans isolate MSU-I
viable yeast was (69.6:3.9), formalized yeast was
(66.7:3.9), car was (22313.1), FHA was (14.2593), Con-A
was (18.6:1.9), and A-DM was (20.1:2.3). Adherence seen
with formalized and viable yeast for MSU-I and other
Candida albicans isolates for each organism was similar
(see Table 1 for statistical data). Decreases in adherence
seen with CY, Con-A, A-DM treatment methods were similar
within treatment groups for MSU-I and other organisms
tested (Figure 15, Table I), and FHA treatment decreased
adherence to the greatest extent for all isolates tested
(Figure 15, Table I).

Figure 16 represents a comparison of the effect of the
treatment methods explained previously on adherence of
W glabrata strains. 9:- mum. 9.-
naransilgsis. s:- M115. and 9.- Wm
pooled human buccal epithelial cells. For M

8()

 

 

fable 1. Comparison of the effect of treatment methods including formalization of the
yeast, cell wall mannan removal, mannan inhibition, and incubation of a
complementary sugar of mannan in the adherence assay, on the adherence of
isolates of Candida um. other Candida 899-. and mm mm to
human buccal epithelial cells.
treatment Methods (x 3 80)
me: n“ v1” 01‘ FM“ Con-A° A-DH‘L
ns
ISU-l 66.713.9 69.613.9 22.3:3.1 16.212.3 18.611.9 20.112.3
(59-72) (62-76) (15-28) (9-17) (15-22) (16-26)
10-16-35F 63.912.6 66.817.1 26.1:2.3 18.611.3 22.911.7 22.8:1.8
(60-69) (66-69) (21-28) (16-22) (20-26) (20-26)
one-1136 58.936.2 61.9;6.1 23.113.6 17.2:2.2 22.8:3.6 22.212.9
(51-66) (52-66) (17-29) (13-21) (16-27) (17-26)
DA-05900 55.816.0 59.613.8 35.2:3.9 27.5:3.9 32.613.8 33.913.8
(67-61) (51-65) (27-60) (19-32) (26-39) (ZS-37)
UCLA 65.512.5 69.8;3.5 21.6:1.6 15.811.1 21.011.9 21.711.6
(60-69) (61-56) (18-26) (16-17) (18-26) (20-26)
1860 37.712.9 61.2:3.1 9.612.5 7.311.8 10.5:1.9 10.6:1.9
(32-62) (35-66) (5-16) (6-10) (7-16) (8-16)
W
16-1 28.311.7 31.110.9 25.8;1.7 18.111.8 26.911.7 26.711.6
(27-31) (30-33) (22-28) (15-20) (21-28) (22-28)
16-3 29.636.9 3S.2:O.9 27.611.6 18.831.3 26.6;1.1 26.8:6.1
(29-33) (36-37) (26-30) (16-21) (26-28) (26-29)
Other Qinglg; sp.
C. stellatoidea 26.511.0 25.810.8 21.011.2 16.310.9 20.911.0 21.131.6
(22-26) (ZS-27) (19-23) (13-16) (19-22) (19-23)
C. parapsilosis 20.510.5 22.0:0.9 18.3:0.9 13.710.9 18.5;0.7 18.311.6
(20-21) (20-23) (17-20) (12-15) (18-20) (17-21)
C. tropicalis 15.510.5 16.930.7 16.6:0.5 10.130.7 13.712.8 16.5:0.8
(15-16) (16-18) (16-15) (9-11) (16-15) (13-16)
C. pseudo-
tropicalis 17.710.7 19.711.0 16.2:0.8 13.311.0 16.210.6 15.510.8
(17-19) (18-21) (15-18) (12-15) (15°17) (16-17)

 

Formal in treated yeast:
bViable yeast.

cCitric acid treated yeast:
dFilamentous Hemagglutinin treated yeast (Hannan inhibition).
'Concanavalin-A treated yeast (Hannah inhibition).
fa-o methylmannopyranoside adherence treatment (complementary sugar).

Hannah removal.

treatment methods and effect kills organism.

FIGURE 16:

Comparison of effects of treatment
methods on adherence of isolates of
Torulopsis glabrata and Candida spp.
(non-C. albicans) to human buccal
epithelial cells. Organisms include:

Iorulopsis glabrata #1
Iorulopsis glabrata #3
Qandida stellatoidea
Candida parapsilosis
Candida tropicalis
Qandiga pseudotzopicalis

a Treatment methods include:

+OK'50

FY: formalin treated yeast (kills

yeast)

CY: yeast treated with citric acid

(decreases cell wall mannan)
VY: viable yeast
FHA: filamentous hemagglutinin
treated yeast (mannan
inhibition)
Con-A: concanavalin-A treated yeast
(mannan inhibition)

A-DM: a-D-methylmannopyranoside
treated adherence mixtures
(complementary sugar mannan
inhibition)

NOZNNMZCU>

60-7

351

30-—

25.—

20-

15-

10..

 

82

 

:3.—

TREATMENT METHODS

FHA

CON-A

83

gm isolate #1, FHA treatment of yeast cells
(18.1-51.8) decreased adherence to the greatest extent as
compared to Con-A (24311.7), A-DM (24311.6), or CY
(25.811.7) samples, which decreased adherence approximately
equally within trials, while adherence of viable yeast
(31.0:0.9) differed little from formalized yeast (28.3:1.7)
(see Table 1 for statistical data). Similar results were
obtained for other organisms tested (Table 1).

Table 1 shows the statistical data for Figures 15-16,
and indicates the average adherence for the treatment
methods of each organism, standard deviations, and ranges
for each particular treatment test. The ranges for specific
tests in some instances are broad, although standard
deviation values are not, indicating that even though a
significant difference exists numerically between highest
and lowest samples, variation between individual readings
was not significant.

The comparison of the remaining relative percentage
of adhering yeast to human buccal epithelial cells after
various treatment methods is shown in Table 2. The
treatment of yeast with a citric acid extraction procedure
decreased adherence of Candida W isolates (range:
23.3-59.3%) much greater than W15 QM strains
(77.8-82.9%) or other gandida spp. tested (81.6-85.2%).

FHA (NJ-66.3%) and Con-A (26.7-54.9%) pretreatment of

84

 

 

Table 2. Comparison of the remaining relative percentage
of adhering yeast to human buccal epithelial
cells after various treatment methods
(treatment/viable yeast x 100)

Treatment Methods (x 1 SD)
finhigct CY FHA, Con-A A-QM_,
W
MSU-I 32.0 20.4 26.7 28.9
10-16-35F 37.2 28.4 35.3 35.2
ONC-1134 37.3 27.8 36.8 35.9
DA-05900 59.3 46.3 54.9 57.1
UCLA 43.0 31.7 42.2 43.6
1840 23.3 17.7 25.5 25.2
W
TG-l 82.9 58.2 80.1 79.4
TG-3 77.8 53.4 75.0 70.5
other Candida sp-
C. stellatoidea 81.6 55.4 81.0 81.8
C. parapsilosis 83.2 62.3 84.1 83.2
C. tropicalis 85.2 59.8 81.1 85.8
C. pseudo-
M 82...: 67.5 82.12 18.7

 

 

 

85

‘yeast isolates, and A-DH (25.2-57.1%) treatment of
adherence mixtures also decreased adherence of .dandida
albigang isolates greater’ than adherence of the other
organisms tested, with ranges of adherence for each
treatment. being 77.8-85.2% for’ CY, 53.4-67.5%' for' FHA,
75.0-84.1% for Con-A, and 70.5-85.5% for A-DH (see Table 2
for results for each organism). Comparison of percent germ
tube formation (Figure 1) and remaining adherence after
treatment methods (Table 2) for Q. albidang isolates
indicates that there is no correlation between ability to
produce germ tubes and degree of decreased adherence seen
using treatment methods which inhibited mannan, as would be
expected if ability to adhere is related to ability to
produce germ tubes. For example, 1180-1 (99% germ tube
formation) CY remaining adherence (32%) was greater than CY
remaining adherence (23.3%) for isolate 1840 (Table 2).
Similar results were found with FHA, Con-A, and, A-DM

treatment methods (Table 2).

DISCUSSION

Adherence of microorganisms to biological surfaces is
considered to be an initial and essential stage in
colonization and infection of the host (Beachey et al.,
1981)- Candida. albicans. other Candidmu and Mia
(Candida) glabgaga are the causative agents of candidiasis,
a primary or secondary opportunistic infection of host
epithelial cell surfaces caused by a member of the genus
Candida (Rippon, 1980). Candida alpjdang is the most
frequently isolated member of the genus in human patient
populations (Rippon, 1980), although in specific disease
states, other Candida sp. are more commonly seen such as in
infective endocarditis of fungal origin where C.
W is most frequently found (Rippon, 1980).
Disease states caused by different Candida sp. are
clinically indistinguishable and affected areas must be
cultured to determine the causative agent of the disease
process (Braunwald, et al., 1987).

Characterization of environmental, developmental, and
growth factors which optimize adherence of C. am to
biological surfaces has been studied extensively. Organisms
in the stationary phase of growth have been found to adhere
in greater numbers than organisms in the logarhythmic phase
of development (King et al., 1980). Many in £13322 studies
have shown that the germinated form of C. 3.1m. an
intermediate form between the blastospore and filamentous

86

87

stage of development, adheres in greater numbers than non-
germinated forms (Kimura and Pearsall, 1978,1980: King et
al., 1980; Sandin and Rogers, 1982: Rotresen et al., 1986).
Although both blastoconidial and mycelial forms of C.
albidang can adhere, invade, and proliferate in an infected
host (Shepherd, 1985), another study showed that the
capacity of C. alpigang to produce hyphae appeared to be an
important but nonessential virulence factor in the
pathogenesis of candidal vaginitis in 1139 (Sobel et al.,
1984).

Preincubation of C. albidadg isolates in tissue
culture medium M-199 prior to use in the adherence assay
favors germ tube formation (Kimura and Pearsall, 1978, 1980;
Sandin and Rogers, 1982). Researchers have postulated that
changes in the cell wall of C. m as it germinates
could be responsible for the increased adherence seen
(Kimura. and. Pearsall, 1978; Sobel et. al., 1981). ‘With
increasing germ tube formation capability for C. aldidang
isolates, increased adherence is seen (Figure 1) . All C.
albiCanfi isolates used in this study were of clinical origin
(Appendix A-I) and were in the stationary phase of
development prior to being subjected to conditions
permitting germination to occur. This study indicates that

for C. W isolates, increased germ tube production

88

tubes results in increased ability to adhere to human buccal
epithelial cells.

Adherence of Candida sp. to biological surfaces is
important in establishment of colonization and infection of
the host (Beachey et al., 1981), for without an effective
means of adhesion to host epithelial surfaces, bodily
secretions, peristalsis, or mechanical trauma would result
in dislodgement of the organisms from affected tissues (King
et al., 1980). C. amima has been found to adhere in
greater numbers than C. ageiiagdidea, C. Cradidaiig, and
other Candida sp. to vaginal cells (King et al., 1980),
buccal cells (Kimura and Pearsall, 1980: King et al., 1980:
Rotresen et al., 1986) and to fibrin platelet matrixes
formed in giggg (Rotresen et al., 1985). Candida ainigana
also adheres more strongly to epithelial cells than fungal
cells. of other Candida sp. (Macura, 1985). Results
indicate that C. ainigang isolate MSU-I adheres to a greater
extent than I. diagraCa strains and other Candida sp. tested
(Figure 2) . I. diagram isolates adhered greater than (in
decreasing order of adherence) C. W, C.
W. C. W. and 9- Wide;
(Figure 2) . These results are in agreement with published
data and parallel the heirarchy of clinical isolates of
Candida obtained obtained from patient specimens (Rippon,
1980). The C. amidang isolate with the least germ tube

89

formation capability (1840) adhered greater than I. giannaga
strains or other non-C. ainiCana sp. tested (comparison of
Figures 1 and 2) . This data supports the concept that C.
aihiCana isolates adhere to a greater extent to human buccal
epithelial cells than other Candida spp. and I. glabgaga,
even when germ tube production is low, and is also in
agreement with published data supporting the concept that
the germ tube is important in adherence of C. ainidana
isolates to human buccal epithelial cells.

It is generally agreed that there is great variation in
the number of receptor sites for C. ainiCana on epithelial
cells collected from different persons (King et al., 1980:
Sandin et al., 1987), although in a study by Cox, 1983,
adherence of C. ainidana to human buccal epithelial cells
was found to be the same in normal adults and children,
suggesting that a stable cell receptor system in present
which is not age dependent. Researchers have hypothesized
that indigenous flora could suppress adherence of C.
ainigang by competing with it for receptor sites on
epithelial cells, modifying these sites to hamper candidal
adherence, or enzymatically altering the yeast surface
(Liljemark and Gibbons, 1973). Specific host defense
factors include cell mediated and humoral immunity as well
as non-specific immune mechanisms which may influence

adherence (Rogers and Balish, 1980) . Non-specific host

90

factors include local defense mechanisms and environmental
conditions (Rippon, 1980). Other factors which might affect
adherence of Candida spp. to epithelial cells include stage
of development and other characteristics of epithelial
cells. Scanning electron microscopy studies have shown non-
uniform distribution of adhering microorganisms with
diminished adherence in areas of active mitosis and
proliferation, and increased adherence to mature flat cells
often in the process of desquamation in studies of adherence
of C. ainigang to vaginal epithelial cells (Sobel et al.,
1982). For C. ainidang isolates, I. gianzana isolates, and
other Candida, sp. tested, variation existed between
individuals in. terms of adherence of 'viable or ‘treated
organisms to human buccal epithelial cells (Figures 3-14),
but that variation was not statistically significant in most
cases (see Appendix C for ANOVA analysis data, and Appendix
B for individual trial results). One particular individual
(male, age 31: position 5 in Figures 3-10) in many cases
differed significantly from the other five individuals
tested, and this can possibly be attributed to the fact that
he had worked with C. aibiCang and other Candida spp. for a
considerable period of time prior to use of his buccal cell
samples in adherence assays. Specific host defense factors
such. as immune :mechanisms could. also account for ‘these

results. Possible explanations for the non-variation

91

between individuals in adherence of particular yeasts,
treated or untreated, to buccal epithelial cells is that the
cells were collected at approximately the same time on days
of experiments, buccal cell donors were candidal free as
determined by oropharyngeal culture, donors were not
receiving antibiotics or antifungal agents at the time.
Other experimental conditions which may have affected
adherence include that buccal cell specimens were processed
immediately upon collection and placed on ice until use
(less than two hours), buccal cell specimens were used for
only one set of experiments and not held over for future use
in adherence experiments, and buccal cells for each
individual were resuspended after processing according to
viable cell count as determined by methylene Blue staining
and hemocytometer count. The stable cell receptor system
for adherence of C. ainidang suggested by Cox, 1983, could
also account for these observations. Significant variation
did exist in terms of number of yeast adhering to individual
buccal cells in each particular test which supports pub-
lished data (Sandin et al., 1987), although specific
numbers of yeast adhering to individual buccal cells in each
trial were not recorded. Stages of development of individ-
ual buccal cells collected from donors were not determined,

which may have had an effect on adherence seen.

92

The study of adherence of C. ainidana to biological
surfaces has included both viable and formalized cells in
different growth stages. Kimura and Pearsall, 1978, found
that once germinated, C. ainiCana cells could be killed in
formalin with no significant decrease in adherence, while
treatment before germination decreased adherence. In a
study of attachment to endothelial cells, viable or killed
Candida organisms were enveloped by membrane processes from
endothelial cell surfaces and were incorporated into the
endothelial cells in phagosomes (Rotresen et al., 1985) .
For C. aipigana isolates, no significant difference was
found between adherence of viable or formalized germinated
yeast specimens (Figures 3-8, Appendix C) . No significant
difference between formalized and viable yeast (blastospore
form) is present for I. glanraga isolates tested or for
other non-C. ainicana Candida spp. tested (Figures 9-14,
Appendix C). Similar results were seen using pooled buccal
epithelial cells for all organisms (Figures 15 and 16, Table
1). Non-C. ainigana organisms were used in the blastospore
stage of development, and C, ainidana isolates were in the
germinated form. Studies were not performed to determine if
germination of these organisms increased adherence ability,
or if formalization before germination of non-C. ainigana

organisms decreased adherence.

93

Characterization of the adhesin which mediates
adherence of C. ainiCana to biological surfaces has received
much attention. The cell wall of C. ainiCana is composed of
chitin, mannan, glucan, and other chemical constituents
including protein (SanBlas, 1982). Five to eight layers of
the cell wall of C. ainidana have been identified depending
on growth conditions or cytochemical techniques utilized
(Poulain et al., 1978), with chitin identified as the
compound composing the bulk of the most electron transparent
layer in transmission electron microscopy studies of germ
tube formation (Cassone et al., 1973). The localization of
mannan in the cell wall of C. ainidana has been confirmed by
Djaczenko and Cassone, 1971: Poulain et al., 1978: and Evron
and Drewe, 1984. Studies by Kimura and Pearsall, 1978,
showed that enhanced adherence of C. ainidana after
incubation in saliva was related to changes in the fungus
itself, and Sobel et al., 1981, found that adherence of C.
ainidana was enhanced by a surface component of the
germinated yeast, postulated to be a glycoprotein. Cassone
et al., 1978, found that mannan is the major constituent of
the fibrillar-floccular layer of the cell wall of C.
ainigana using concanavalinsA. POpe and Cole, 1981, found
that yeast were associated with a mucus layer on epithelial
surfaces throughout the gastrointestinal tract in scanning

electron microscopy studies, and a study by Tronchin et al.,

94

1984, indicated that attachment of C. ainigana to buccal
epithelial cells appeared to involve spatial rearrangement
of their cell wall surface through development of a
fibrogranular surface layer which was detected using
specific carbohydrate staining techniques and concanavalin-A
binding, indicating that attachment of yeast to epithelial
cells was mediated by a mannose receptor. Similar studies
have not been completed for non-C. ainiCana Candida sp.
Inhibition of adherence of C. ainigana to different
epithelial cell surfaces has been accomplished through the
use of many different and varied compounds. Pretreatment of
C. aipiCana with trypsin, chymotrypsin, or proteinase
decreased adherence significantly (King et al., 1980: Maisch
and Calderone, 1980). Loss of adherence after treatment of
yeast cells with a-mannosidase or papain suggests that the
cell wall mannoprotein is an essential component of the C.
ainigana adhesin (Lee and King, 1983). Concanavalin-A (con-
A) pretreatment of C. ainidans inhibited adherence (Sandin
and Rogers, 1982) , and addition of a—D-meh tylmannopyrano-
side in the adherence assay also inhibited adherence (Sandin
et al., 1982) . Studies of controlled degradation of the
cell surface of C. ainidana indicated that mannan and manno—
proteins appeared to be important constituents of the
adhesin mediating adherence (Rotresen et al. , 1986) . The

effect of 'various treatment. methods on adherence. of (C.

95

ainigana isolates to pooled (Figure 15) and individual
(Figures 3-8) human buccal epithelial cell samples using a
citric acid (CY) extraction technique which decreased cell
wall mannan (Tronchin et al., 1984), cell wall mannan ligand
inhibition with the use of con-A and filamentous hemag-
glutinin (FHA), a purified protein adhesin of W
nangnaaig which binds mannan and mediates adherence of the
organism to ciliated respiratory epithelial cells (Sato et
al., 1984), and a-D-methylmannopyranoside treatment of
adherence mixtures, which is a complementary sugar of mannan
that inhibits adherence of C. ainigana isolates to human
buccal epithelial cells (Sandin et al., 1982) showed that
all treatment methods decreased adherence of C. aim
isolates to human buccal epithelial cells. Filamentous
hemagglutinin treatment decreased adherence to the greatest
extent. Little variation between adherence of organisms
(viable or treated) to individual (Figures 3-8) or pooled
(Figure 15) buccal cells ‘was seen. 'Wide 'variation in
remaining adherence for each treatment method between C.
ainigana isolates was noted (Table 2). Comparison of
remaining adherence after treatment methods (Table 2) to
percent germination of these isolates (Figure 1) reveals no
correlation of these factors. The degree of inhibition of
adherence seen with FHA and Con-A, decreasing cell wall

mannan (CY), or complementary sugar inhibition (A-DM) is not

96

dependent on germ tube formation capability, indicating that
differences in cell wall mannan concentration and structure
could be present in different isolates of germinated C.
ainidana. These results indirectly support the concept that
changes in the cell wall of C. ainidana as it germinates are
responsible for the increased adherence seen (Kimura and
Pearsall, 1978, 1980: Sobel et al., 1981), although the lack
of correlation between germ tube formation capability and
inhibition of adherence suggests that other factors such as
growth conditions or cell wall differences for different
strains of C. ainiCana may be playing a role in adherence
capacity of individual organisms.

The effect of treatment methods on the adherence of I.
giannaia isolates and Candida spp. other than C. aipigana
to individual (Figures 9-14) and pooled (Figure 16) human
buccal epithelial cells indicates that all treatment methods
inhibit adherence. Filamentous hemagglutinin treatment had
the most dramatic effect, decreasing adherence to the
greatest extent. Changes in adherence to individual or
pooled buccal cell preparations using the treatment methods
indicated were significant from viable yeast preparations
(see ANOVA analysis results for Figures 9-14 in Appendix C),
although the changes in adherence seen between CY, con-A,
and A-DH treated yeast for each organism were minimal and

and less than that of FHA. No differences in adherence

97

inhibition were noted when comparing adherence of organisms
to individual or pooled buccal epithelial cells. Little
variation in remaining adherence between non-C. alnigang
Candida spp. or I. diannana strains within trials for a
particular treatment method were noted (Table 2) . This
suggests that differences in mannan mediated adherence for
these organisms are small, and that other mechanisms of
attachment using other adhesin systems could be operational
for these organisms.

The ability of FHA to inhibit adherence greater than
other mannan inhibitors or other treatment methods for C.
alnidana isolates (Figures 3-8 and 15), and for non-C.
ainidana organisms (Figures 9-14 and 16) was demonstrated.
Filamentous hemagglutinin is a protein secretion of
Bazdanaiia M which mediates adherence of the
organism to human 'ciliated respiratory epithelial cells in
21329 (Tuomanen and Hendley, 1983: Urisu et al., 1986). FHA
was originally thought to be a component of the fimbriae of
3. mid (Sato et al., 1984) which was identical to
hemagglutinating proteins present in the fimbriae of other
piliated bacteria which mediated adherence or these
organisms to biological surfaces (Korhonen et al., 1982) ,
but it is now believed to be an extracellular protein
adhesin secretion of the organism (Zhang et al., 1985). FHA

is a large protein with a molecular weight approaching 106

98

(Urisi et al., 1986). It has been shown to enhance the

adherence of Hangnniina infinanza Type B and inhibit the

adherence of 55.114221252993311: nndnndniaa to human respiratory
epithelial cells in yinzg, suggesting that piracy of

adhesins may contribute to superinfection in mucosal
diseases, although it was impossible to determine if the
interaction between heterologous bacteria and FHA was
receptor mediated or nonspecific (Tuomanen, 1986) . The
potential pathophysiological importance of this interaction
is suggested by the idea that piracy of adhesins could allow
or enhance adherence of organisms to surfaces, or inhibit
the adherence of other organisms to specific biological
surfaces. The interaction seen between FHA and organisms
(Seen in Figures 3-8 and 15 for C. ainiCana, and Figures 9-
14 and 16 for non-C. ainidana isolates) indicates that the
binding of this protein to yeast (germinated C. ainigang
isolates) decreases adherence ability. The FHA used in this
study was secreted by 3. Mia, but similar compounds
are secreted by other piliated strains of bacteria
(Korhonen et al., 1982). In addition, it has been shown

that the presence of certain piliated strains of bacteria

such as 31.228.151.13 pnanngniaa (Centeno et al., 1983) and

W Cali (Makrides and MacFarlane, 1983) can
increase Candida adherence to epithelial cells. It is

recognized also that indigenous microflora of specific site

99

areas in humans can suppress the adherence of C. ainidana,
such as in the gastrointestinal tract (Rippon, 1980).
Treatment of humans with antibiotics, antimetabolites, and
corticosteroids disturbs the normal flora distribution in
humans and predisposes them to opportunistic invasion by
Candida sp. (Braunswald et al., 1987). Other disease
states, metabolic abnormalities, and immunosuppression, are
factors which have also been found to predispose patients to
candidiasis (Rippon 1980). The piracy of adhesins secreted
by remaining flora and other microorganisms in such situ-
ations could possibly help explain and partially account
for the increased incidence of candidiasis seen clinically.
The results presented in Figures 3-16 regarding FHA support
the concepts of adhesin piracy in 111.110.. and indirectly
demonstrate the ability of microorganisms to affect the
adherence of other microbials to biological surfaces.
Because the effect of FHA on adherence was greater than that
of the other treatment methods which decreased adherence
through mannan inhibition or decreased cell wall mannan for
all organisms, the results suggest that adherence may be
mediated by other factors in addition to mannan receptor
function in Candida spp. and I. gianraga. Comparative
studies were not performed which would have quantified the
cell wall mannan removed with the citric acid extraction

technique for each organism, nor were studies done to

100

determine if all mannan receptor sites were inhibited or
blocked with FHA, con-A, or a-D-methylmannopyranoside.
Titration assays were performed to determine the maximum
inhibition of adherence possible using the methods indicated
for a representative organism (C. ainigana isolate MSU-I) .
The relative percent remaining adherence after
treatment methods for all organisms tested showed that
variation for all treatment methods exists for C. 913%
isolates, but the change in adherence for other organisms
used is relatively constant within a particular treatment
method (Table 2) . CY, con-A, and A-DM decreased adherence
approximately equally within tests for one organism, which
is different than the effect of these treatment methods on
adherence of C. ainidana isolates seen also in Table 2.
Percent adherence of C. ainigana isolates after subjection
to treatment methods was less than remaining adherence for
all other organisms tested. Possible reasons for these
observations include differences in cell wall structure and
mannan concentration between different Candida sp. and
within the C. ainidana species which could affect the
characteristics of the adhesins responsible for adherence of
particular organisms. Since only C. ainidana isolates were
in the germinated form in the adherence assays, the dif-
ferences in adherence seen with the treatment methods used

for C. ainidana, and the differences noted between non-C.

100

determine if all mannan receptor sites were inhibited or
blocked with FHA, con-A, or a-D-methylmannopyranoside.
Titration assays were performed to determine the maximum
inhibition of adherence possible using the methods indicated
for a representative organism (C. M isolate MSU-I) .
The relative percent remaining adherence after
treatment methods for all organisms tested showed that
variation for all treatment methods exists for C. ainidarns
isolates, but the change in adherence for other organisms
used is relatively constant within a particular treatment
method (Table 2) . CY, con-A, and A-DM decreased adherence
approximately equally within tests for one organism, which
is different than the effect of these treatment methods on
adherence of C. albicans; isolates seen also in Table 2.
Percent adherence of C. ainidana isolates after subjection
to treatment methods was less than remaining adherence for
all other organisms tested. Possible reasons for these
observations include differences in cell wall structure and
mannan concentration between different Candida sp. and
within the C. ainiCana species which could affect the
characteristics of the adhesins responsible for adherence of
particular organisms. Since only C. alnigana isolates were
in the germinated form in the adherence assays, the dif-
ferences in adherence seen with the treatment methods used

for C. ainidana, and the differences noted between non-C.

101

ainidana isolates and I. giannaga strains as a group
compared to C. ainigana could be related to the germination
process in addition to cell wall structural differences, and

possible differences in adhesin characteristics.

APPENDIX A

102

APPENDIX A-I

Source of organisms used in studies:

 

 

QBEAHIEH £99322
Candida alniaana

HSU-I Fecal specimen isolate collected by
Linda Olsen, MSU Med. Hyc. Lab.

10-16-35F Vaginal isolate from Sparrow Hosp.,
Lansing, MI.

ONC-1134 Vaginal isolate from Olin Health
Center, HSU.

DA-05900 Vaginitis case isolate from Dr.
Cooper's lab., Dallas, TX.

UCLA Vaginal isolate from Dr. Howard's
lab., UCLA.

1840 Recurrent vaginitis case isolate from

Dr. Magee's lab., MSU.

Taralanaia glabrata

1 Vaginal isolate from Lansing General
Hosp., Lansing, MI.

3 Vaginal isolate from Sparrow Hosp.,
Lansing, MI.

Other Candida spp-

C. stellatoidea Vaginal isolate from CDC Proficiency
Test.

C. parapsilosis Vaginal isolate from CAP Proficiency
Test.

C. tropicalis Vaginal isolate from Sparrow Hosp.,

Lansing, MI.

C. pseudotrop- Vaginal isolate from Med. Hyc. Lab.,
icalis Duke Univ.

103

APPENDIX A-II

Organisms

Sabouraud's Dextrose Agar

 

Transfer loop of cells to 100 ml
Trypticase Soy Broth + 4% glucose

rotary shaker,

Stationary Growth Phase

 

 

 

 

 

 

24-48 hrs., 37*C

15 hrs., 180 rpm,
37*C

Candida alhidana Other Candida sp. ,
l Inmlanaia alahmfa

M-199 Tissue Culture Medium

1 hr., 37°C, 180 rpm

rotary shaker Methylene Blue
Viability Count
Wash 3X in PBS
Resuspend in PBS
Count germinated and non-germinated
cells with Methylene Blue staining
to determine viability and % germ
tubes

Resuspend cells to 106 cells/ml PBS

Resuspend 1/8 of cell solution Viable Yeast (VY)

in 0.5% formaldehyde in PBS
for 30 min. at 4°C

Wash 3X in PBS
Resuspend cells to 106 cells/ml PBS

Formalized Yeast (FY)

104

APPENDIX A-III

ADHERENCE ASSAY

 

0.2 ml yeast 12 x 75 mm test tubes
(3 for each test)

 

 

0.2 ml buccal cells-—-

rotary shaker, 180 rpm,
1 hr., 37°C

 

 

filter each tube on a L
polycarbonate filter (12 um)

wash with 100 ml PBS
(continual agitation)

 

Stain filters with Gram's Crystal Violet

 

Count number of yeast adhering
to 100 buccal cells
(430x, light microscopy)

* Double blind conditions were used in all studies

* a-D—methylmannopyranoside added after buccal cells
and yeast were pipetted into test tubes

105

APPENDIX A-IV
TREATMENT METHODS

Viable Yeast

Treatment] Methods

 

CY Con-A

5.0 ml VY 5.0 m1 VY
Resuspend in 15 ml Resuspend in 15 ml
0.1 M Citric Acid 10 ug/ml Con-A

in PBS in PBS
Autoclave 1 hr. 45 min., rotary

at 120°C shaker, 180 rpm

Room Temp.

Wash 3X in PBS

Wash 3X in PBS

Resuspend to 5.0 ml

PBS

 

Resuspend to 5.0 ml
PBS

“I“
5.0 ml VY
Resuspend
in 15 ml
40 ug/ml
FHA in PBS

45 min.,
rotary
shaker,
180 rpm,
Room Temp.

Wash 3X in
PBS

Resuspend
to 5.0 ml
PBS

 

 

Proceed with Adherence Assay

a-D-methylmannopyranoside was added to three adherence
test tubes containing 0.2 ml viable yeast and 0.2 ml
buccal cells as an adherence treatment method.

Formalized yeast were also used in adherence assays.

APPENDIX B

 

106

APPENDIX 8

clinical isolates of Candida ainiaana with differing germ-tube formation ability to

Comparison of the effect of different treatment methods on adherence» of six
six different human buccal epithelial cell samples.

1.

ISU-l -- 99X Germ tubes

Organism:

 

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Organism:

 

(n + 59)

CA'DH

 

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107

clinical isolates of Candida albigana with differing germ-tube formation ability

Comparison of the effect of different treatment methods on adherence» of six
to six different human buccal epithelial cell samples, cont'd.

UCLA -- 52% Germ tubes

Organism:

 

trgatmeng Methods (x + §92

5:0"

Con-A

 

FHA

CY

VY

FY

 

 

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Comparison of the effect of different treatment methods on adherence of two
cell samples.

torulopsis glabrata 1

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treatment methods and effect kills organism.

iable yeast.

 

'Formal in treated yeast

by

c
d
e
f

Mannan removal.
Filamentous Hemagglutinin treated yeast (Wannan inhibition).

Concanavalin-A treated yeast (Hannah inhibition).

a-O methylmannopyranoside adherence treatment (complementary sugar).

Citric acid treated yeast

APPENDIX C

109

APPENDIX C

ANOVA results of statistical analysis of data:

1. Wings Bergen

LRH
ALR
A0
EB
MK
LS

mmthH

II. Organism: Graph # = Figure #

1............3
2............4
3............5
4............6
5............7

6IIIIIIIIIIII8

7............9

8............10

9............11
10............12
11............13
12............14

GENERALIZATIONS
EAQIQB_A=
Tests 1-6:

Tests 7-12:

EAQIQB_B=

110

FY and VY significantly differed from the
rest except each other.

FHA significantly differed from the rest
in many cases.

MK is significantly different in all cases where a
significant difference exists in FACTOR B.

RESULTS OF GRAPH #1

FACTOR A
FY significantly differs from rest except VY
VY significantly differs from rest except FY
CY ALR significantly differs from FHA ALR
AD significantly differs from A0
EB significantly differs from EB
FHA A0 significantly differs from a-DM A0
FACTOR B
FY A0 significantly differs from FY MK

WITHIN SUBGROUPS

FY AO significantly differs from All except VY

RESULTS OF GRAPH #2

FACTOR A

FY significantly differs from all but VY
VY significantly differs from all but FY

 

111

FACTOR B

VY MK significantly differs from VY LS

RESULTS OF GRAPH #3

FACTOR A
FY significantly differs from all but VY
VY significantly differs from all but FY
CY LRH significantly differs from FHA LRH
FACTOR B
FY LRH significantly differs from FY MK
CY ALR significantly differs from CY MK
VY MK signficantly differs from All VY except
LS and A0
FMA MK significantly differs from FMA ALR
22
ConA ALR significantly differs from ConA MK
a-DM MK significantly differs from a-DM ALR
23.
RESULTS OF GRAPH #4
FACTOR A
FY significantly differs from All except VY
VY significantly differs from All except FY
FACTOR B

FY ALR significantly differs from FY MK

 

 

RESULTS OF GRAPH #5

112

differs
differs

differs

from
from

from

All except VY
All except FY

FHA LAS

differs
differs

differs

differs

from
from

from

All except VY
All except FY

VY ESB

VY AO
ESB

FACTOR A
FY significantly
VY significantly
CY LAS significantly
RESULTS OF GRAPH #6
FACTOR A
FY significantly
VY significantly
FY ESB significantly
FACTOR B
VY MAL significantly
RESULTS OF GRAPH #7
FACTOR A
FY significantly
VY significantly
VY LRH significantly
FHA ALR significantly
A0 significantly
ESB significantly
MAK significantly
LAS significantly
VY LAS significantly

differs
differs

differs
differs
differs
differs

differs
differs

differs

from
from

from
from
from
from

from
from

from

FHA
FHA

ConA LRH

FY ALR, CY ALR
FY A0, ConA A0
All ESB except
ConA

All MAK

All LAS

a-DM LAS

RESULTS OF GRAPH #8

FACTOR A
FHA significantly
VY significantly

113

differs
differs

from

from

All

All except FY

RESULTS OF GRAPH #9

FACTOR A
FHA significantly
VY ALR significantly
A0 significantly

differs

differs

differs

All

ConA ALR, a-DM
ALR CY A0, a-DM
A0

a—DM A0

RESULTS OF GRAPH #10

FACTOR A
FY significantly
VY significantly
FHA LRH significantly
significantly
significantly
FHA A0 significantly
significantly
significantly
FHA LS significantly
significantly

differs
differs

differs
differs
differs

differs
differs
differs

differs
differs

from
from

from
from
from

from
from
from

from
from

FHA
FHA

CY LRH
ConA LRH
a-DM LRH

CY AO
ConA A0
a-DM A0

CY LS
ConA LS

RESULTS OF GRAPH #11
FACTOR A

FHA significantly

differs

114

RESULTS OF GRAPH #12

FACTOR A

VY

FY LRH

VY AO

FY AO

VY LRH

significantly differs
significantly differs
significantly differs
significantly differs
significantly differs

significantly differs

from
from
from
from
from

from

FHA LRH

ConA AO,
a-DM AO

FHA AO

a-DM LRH

 

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