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This is to certify that the

 

 

 

 

“i
thesis entitled
Estrogen Actions on Trichomonas
vaginalis, Tritrichomonas foetus,
and Trichomonas gallinae
J presented by

Nancy L. Mummaw

has been accepted towards fulfillment
of the requirements for

Master Of Science degree in Medical Technology

 

 

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Major professor

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.-—-——-———

Estrogen Actions on Trichomonas vaginalis,

Tritrichomonas foetus and Trichomonas gallinae

By

Nancy L. Mummaw

A THESIS

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

MASTER OF SCIENCE

Department of Medical Technology

1989

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(.2)

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ABSTRACT
Estrogen Actions on Trichomonas vaginalis,
Tritrichomonas foetus and Trichomonasggallinae
by

Nancy L. Mummaw

The hormonal milieu can alter susceptibility to infection. The effect
of hormones on Trichomonas vaginalis, Tritrichomonas foetus, and
Trichomonas gallinae, was studied using axenically cultured clinical
isolates. Estrogens in physiological concentrations, decreased the
growth of T. vaginalis and adherence to McCoy cells in vitro, and
acted as a chemorepellent. The specificity of these effects was
verified by their being blocked with tamoxifen, an anti-estrogen, by
the dose and time dependency of the responses, and by the lack of
effect with other hormones. T. gallinae and T. foetus were not
responsive to estrogens. Estrogen binding site determinations showed
all isolates displayed specific estrogen binding with similar binding
affinity (Ka, Kd), however there were significant differences in

estrogen binding capacities comparing T. vaginalis with T. gallinae

 

and T. foetus. T. vaginalis displayed significantly greater total
estrogen binding capacity compred with the non-estrogen responsive
trichomonads. These results show that estrogens directly decrease the
virulence of T. vaginails, however, interactions between estrogens and
mammalian cells have a different effect and promote the development of
infection. Complicated interactions between hormones, microorganisms,
and mammalian cells ultimately determine whether such exposure

predisposes to or prevents the development of infection.

ii

ACKNOWLEDGMENTS

I would like to thank my committee members Barrett Sugarman, M.D.
Professor of Medicine, Microbiology and Public Health, Barbara Styrt,
M.D., Associate Professor of Medicine, Microbiology and Public Health,
Martha Mulks, Ph.D., Associate Professor of Microbiology and Public
Health, and Sharon Zablotney, Ph.D., Associate Dean, College of

Natural Science for their assistance and continued support.

iii

TABLE OF CONTENTS

Introduction
Materials and Methods
Trichomonads
Materials
Growth Assay
Radiolabelling
Chemotaxis
Adherence Assay
Preparation of Cytosol
Steroid Binding
Statistical Methods
Results
Growth
Chemotaxis
Adherence
3H 17-B-estradiol binding
Estrogen Binding Affinity and Binding Capacity
Summary and Conclusions
List of References

iv

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Table
Table
Table
Table

Table

LAJNH

LIST OF TABLES

Effect of l7-B-Estradiol on the growth of trichomonads
Effect of hormones on the growth of T. vaginalis
Effect of lOng/ml 17-B-Estradiol on the chemotaxis
of trichomonads
Effect of l7-B-Estradiol on the adherence of
trichomonads
Estrogen binding of trichomonads

 

10
11

14

15
17

Introduction

Trichomonas vaginalis, the causative agent of human genital
trichomoniasis was first described by A. Donne' in 18361. Yet after a
century and a half, many questions regarding the pathogenicity of this
organism remain unanswered. A typical human infection by the parasite
lacks easily discernible features associated with many other
infectious diseases, and symptomless chronic infections are known to
occur. The more subtle characteristics of the disease have not been
studied in an experimental host, because infection corresponding to
that in humans has not been reproduced despite attempts with a number

of animal species.

Trichomonas vaginalis has been cultivated with varied success under
conditions too numerous to cite. None of the culture methods can
sufficiently distinguish between virulent and avirulent strains. The
classification of isolates as pathogenic or nonpathogenic is based
soley on the severity of the symptoms of the patients from whom the
particular strains were recovered. There is no other direct procedure

by which the pathogenicity of the organisms can be determined.

Infection may elicit a profuse, acute, inflammatory discharge
containing many polymorphonuclear neutrophils and trichomonads.
Numerous imidazole compounds have activity against Trichomonas
vaginalis, and occasional reports of resistance to these compounds
(e.g. the S-nitroimidazole metronidazole) explain some cases of

clinical failurez. Because treatment failures seem to be far more

common than the presence of resistant organisms other factors related

to the virulence of these organisms should be considered.

Observations of sex differences in host susceptibility to infectious
agents have prompted numerous studies dealing with the effects of
hormones, particularly estrogens, on infectious disease
processes3’4’5. These studies have clearly indicated that
therapeutic, and in some cases physiological, levels of estrogens
alter host susceptibility to selected microbial agent56’7. Although
the underlying modes of action are not completely understood, many
potential mechanisms by which estrogens may act to alter the host
susceptibility to infection have been identified. One such mechanism
identified was modification of host epithelial cell number,
morphology, or receptor site synthesis on epithelial cellsA’S. Other
studies have suggested estrogen induced immunosuppression of the
immune response. The divergent effects of estrogen on resistance may,
in some instances, be due to the quantity of chemical injected or
route of infectiongrlo. In addition, it is likely that altered host
resistance correlates with immune function disturbances caused by
estrogenic compounds. For example, estrogens are potent stimulants of
the mononuclear phagocytic system but suppress natural killer cell
activity and T cell-mediated immunity as we1111»12’13. Estrogens may
induce these alterations by affecting the production of
immunoregulatory thymic hormoneslA. Increased host resistance to
certain microorganisms is often attributed to estrogen-related
enhancement of macrophage activityls. Conversely, estrogen-induced

defects in natural killer cell functions and T cell responses may

decrease host resistance to other infectious agentslé. Other hormone-
induced changes in host susceptibility to infection are alterations of
host defense factors such as production of cervical mucus in the

female genitourinary tract8.

Receptor proteins have been reported in and are thought to function in
an analogous fashion in simple eukaryotes as in higher organismsl7.
Recent studies of the interactions of mammalian steroid hormones with
fungi, protozoa, and other microorganisms have demonstrated that
hormones may directly alter the virulence of these organisms by
various mechanisms including the ability of the organism to adhere to
host epithelial cells and by directly altering the growth of the
organism18’19. In addition, treatment of fungal cultures with steroid
hormones has resulted in the demonstration of steroid modulated
responses in some pathogenic fungizo. It has been postulated that the
effect of the hormone may be related to the modulation of the

pathogenesis of these fungi21.

Studies have shown infections with Trichomonas vaginalis are more
prevalent after exposure of women to estrogens. In one study the
incidence of T.gyagina1is infections increased from 4% after one month
of oral contraception use to 36% after 3 months and 58% after six
months of use. Women practicing other forms of birth control showed

no increase in T. vaginalis infectionszz. In an attempt to determine

 

the specific actions and possible pathogenic role of hormones,
particularly estrogens, on axenically cultured trichomonads,_in vitro

growth, chemotaxis, adherence, and hormone binding experiments were

performed with Trichomonas vaginalis (the human female genitourinary

 

tract pathogen) Tritrichomonas foetus (a bovine genitourinary
pathogen), and Trichomonas gallinae (a gastrointestinal tract pathogen
of birds). l7-B-estradiol was focused on because it is the
predominant estrogen found in mammals as well as lower species. The

normal human serum level of 17-B-estradiol is 2-3 x 10"10 M.

Materials and Methods

Trichomonads

T. vaginalis isolates were cultured from women who were positive for
T. vaginalis when saline wet mounts were examined by light microscopy.
Sterile cotton swabs were used to inoculate vaginal secretions into 8
x 100 mm borosilicate sterile glass screw-capped tubes containing 6 ml
Diamonds DL8 defined medium pH 7.2, supplemented with 8% (v/v) human
serum, 40 ug/ml gentamicin and 2.5 ug/ml amphotericin B23. Cultures
were maintained in DL8 with AZ (v/v) heat-inactivated equine serum at
37°C in air and were subcultured every three to four days (after
reaching the stationary phase). Antibiotics were removed from the
medium after several passages. Antibiotic-free DL8 medium was used

for all Trichomonad experiments. T. gallinae (ATCC #30227 and #30230,

 

American Type Culture Collection, Rockville, MD) were grown in CTLM
(ATCC medium formulation #745), washed in PBS, and resuspended in DL8
growth media for all assays. Tritrichomonas foetus isolates were
obtained from clinical bovine specimens and were supplied by Dr. Bob
BonDurant at the University of California, Davis. These isolates were
maintained in ATCC medium #745 and were prepared for assays in a

manner similar to the T. gallinae isolates.

 

Materials [3H]- l7-8-estradiol (100 Ci/mmol) in ethanol was purchased
from Research Products International Corp. (Mount Prospect, IL). Non-
labelled hormones (Sigma, St. Louis, MO) and the anti-estrogen
tamoxifen (Stuart Pharmaceuticals, Wilmington,DE) were dissolved
initially in 100% ethanol, and then diluted in PBS, pH 7.3 to yield
stock aliquots containing less than 0.1% (v/v) ethanol at IOOX final
estrogen concentration. Baseline trichomonad medium contained less

than 0.12 of the lowest final estrogen concentration used in assays.

Growth Assay. Cultures of trichomonads were centrifuged at 250x g for
five minutes and resuspended in 10.0 ml of medium. After vortexing,
0.1 ml (approximately 2.5 x 103 organisms) was added to each new tube
containing 6 ml of growth medium with varying concentrations of
hormones and tamoxifen, an anti-estrogen. The tubes were incubated at
37°C for up to 3 days. Tubes were centrifuged, the supernatant
removed, and the trichomonads were resuspended in 1.0 ml PBS and

counted with a hemacytometer.

Radiolabelling. Exponential phase isolates (1-2 days) of trichomonads
in growth medium were radiolabelled with SLICi per ml of [3H-]
thymidine, specific activity of 78.3 Ci/mmol (DuPont, New England
Nuclear Research Products, Boston, MA). Organisms were incubated with
label for 18 hours at 37°C and then were washed 3 times in PBS before

use in assays.

Chemotaxis. Washed, radiolabelled trichomonads were resuspended in
0.5 ml medium to a concentration of 1 x 105 organisms/ml and placed in
the bottom of a 24-well transwell plate (Costar, Cambridge, MA). A
0.45 um pore polycarbonate membrane filter insert was placed on top of
the trichomonad suspension and 0.5 ml medium was added on top of the
membrane. Concentrated estradiol was added to the bottom or the top

medium.

After one hour incubation at 37°C during which the migrating
trichomonads were trapped in the filters, the membrane inserts were
removed, inverted, and blotted to remove excess fluid. The membranes
were removed from the inserts and solubilized with 0.5 ml of 0.5M
protosol (DuPont, NEN Research Products, Boston, MA). Scintillation
fluid was added and radioactivity was assessed in a liquid
scintillation counter. Duplicate experiments were done with known
quantities of trichomonads to allow conversion of counts per minute

(CPM) values to number of trichomonadsza.

Adherence assay. McCoy cells (mouse fibroblasts, not known to respond
to estrogens) were cultivated in 24-well Costar culture plates with
Eagles minimum essential medium with Earles salts (Gibco labs, Grand
Island, NY) plus 10% (v/v) fetal bovine serum (Hyclone Labs, Logan,
UT). Medium was removed from confluent monolayers (containing about 2
x 105 cells/well) and washed radiolabelled trichomonads, previously
incubated for one hour with supplemental estradiol or PBS, were added

at a concentration of 2 x 106 trichomonads in 1.0 ml medium

consisting of 2 parts cell culture medium and 1 part trichomonad

growth medium.

After a 45 minute incubation, non-adherent trichomonads were removed
by rinsing each well 3 times with PBS. The contents of each well
(McCoy cells and adherent trichomonads) were digested with 0.5 ml 0.1
M NaOH for 30 minutes on a rotator. The digest was pH neutralized
with HCl, transferred to scintillation vials containing scintillation
fluid, and radioactivity was measured by counting. CPM were converted

to the number of trichomonads as described earlier.

Preparation of Cytosol. Trichomonads were cultivated in 100 ml of
growth medium to the stationary phase (about 3 days), washed three
times in PBS, and resuspended in 5 ml of 10 mM Tris, 1.5 mM EDTA, 250
mM sucrose, 12 mM monothioglycerol, and 10 mM sodium molybdate buffer
pH 7.4 at 4°C for 1 hour. Trichomonads were disrupted by sonification
in ice by two five second bursts at the lowest setting which
microscopically disrupted the cells. After disruption, large cellular
fragments were removed by centrifugation at 1,000 x g for 10 minutes
at 4°C. This supernatant was ultracentrifuged at 100,000 x g at 4°C
for one hour. Cytosol protein concentrations were determined by the

Bio-Rad Protein Micro Assay Procedure.

Steroid Binding. To determine total binding, 250 ul aliquots of

 

cytosol were incubated with 6 different concentrations of 3H-17-B
estradiol at 4°C for 3 hours (concentrations ranged from 2x10‘10M to

8x10'9M). Hormone-receptor binding, which is saturable, was

distinguished from non-saturable, nonspecific binding by performing
parallel incubations of radioactive hormone in the presence of a vast
excess (ZOO-fold molar excess) of nonradioactive hormone. Such an
excess only minimally occupies the nonspecific sites, and therefore
does not block nonspecific binding; receptors, on the other hand, are
overwhelmingly filled by the excess nonradioactive hormone, and a
negligible amount of radioactive hormone binds to them. Bound hormone
was separated from free hormone by two ultrafiltrations (by
centrifugation) with 10,000 molecular weight cut-off low-protein-
binding cellulosic membrane ultrafiltration units (Millipore, Bedford,
MA). Retentates were counted in a liquid scintillation counter.
Calculated counting efficiency of 3H-estradiol was 32% and background
counts averaging 30-40 CPM were subtracted from all calculations.
Specific binding was calculated by subtracting non-specific binding
from total binding. Standard techniques were used to determine
affinity and dissociation constants (Ka,Kd) and binding capacitiesl7.
Verification of protein in the binding sites was performed by
incubation of the cytosol for 20 min with 100 ug/ml trypsin (Sigma) or
PBS control at 37°C followed by cooling and performance of the binding

assay.

Statistical Methods. All growth, chemotaxis, and adherence
experiments were repeated at least 4 times. Estrogen binding
experiments were all performed at least three times. Results were
analyzed with one- and two- tailed t-tests. Data are expressed as

mean : standard deviation unless otherwise indicated. Large standard

deviations are due to the day to day variation of the growth of the

trichomonads.

Results

Egggth. Numerous T. vaginalis isolates were incubated with hormones
and the growth of the organisms was measured. Only the estrogens had
a significant effect on growth, which was inhibitory. There was no
effect of estradiol on the growth of T. foetus or T. gallinae strains
when compared to T. vaginalis (Table 1). Growth inhibition did not
occur before one day and was maximal after 2-3 days incubation with
estrogens at 1-2 ng/ml (3.7-7.4 x 10'9M). The specificity of this
effect was verified by its concentration dependency. No effect on
growth was noted with concentrations of estradiol less than 1.8x10’
10M. Higher concentrations produced no greater inhibition of growth.
The specificity was further investigated by using the anti-estrogen,
tamoxifen, as an estrogen blocking agent. Tamoxifen alone had no
effect on the growth of the T. vaginalis isolates at concentrations of
up to lOOng/ml; however, at 1ng/m1 it completely blocked the growth
inhibition that occurred in the presence of estrogens (Table 2).
Exposure to estrogens during growth had no effect on the morphology
and motility of trichomonads as observed by 1000x phase-contrast light
microscopy. Scanning electron microscopy of glutaraldehyde-fixed
trichomonads exposed to 3 days of estradiol also failed to show any

morphological effects.

Chemotaxis. The use of a membrane filter separating media with

trichomonads on one side, and various media components (i.e. nutrient

10

Table 1. Effect of 17-B-Estradiol on the growth of trichomonads”

 

Growth+

Isolate Control 17-B-Estradiol (lng/ml)
T. vaginalis-l 1430 i 701 801 1 513*

T. vaginalis-Z 1481 i 612 926 i 523*

T. vaginalis-3 1763 i 1210 1304 i 1207*

T. foetus-1 1280 i 153 1367 i 361

T. foetus - DI/#3 1146 i 201 1194 i 158

T. gallinae - 30227 769 i 478 761 i 476

T. gallinae 30230 893 i 585 797 i 487

# All data expressed as mean 1 standard deviation number of
Trichomonads x 10'3

Growth after 72 hours

Significantly different from control (P<0.05)

+

3(-

11

Table 2. Effect of hormones on the growth of T. vaginalis#

Growth+

Hormone Isolate 1 Isolate 2 Isolate 3
None (PBS control) 1402 i 713. 1431 i 612. 1721 i 1204.
17-B-Estradiol (1ng/ml) 800 i 511" 902 i 507" 1321 i 1200“
17-B-Estradiol (lng/ml
and Tamoxifen (lOng/ml) 1521 i 1120 1431 i 1001 1624 i 902
Tamoxifen (10ng/m1) 1362 i 901 1346 i 1210 1763 i 908
Tamoxifen (100ng/ml) 1411 i 1108 1382 i 960 1631 i 942
Estriol (lng/ml) 913 i 601“ 1101 j: 476 1583 i 1607
Hydrocortisone

(0.4 ug/ml) 1248 i 1163 1071 i 928 1638 i 917
Prednisolone (lug/ml) 1284 i 976 1162 i 714 1638 i 1701
Progesterone (20ng/ml) 1214 i 904 1003 i 713 1648 i 1528
Testosterone (20ng/ml) 1392 i 1128 1375 i 708 1569 i 1249
# All data expressed as mean : standard deviation number of

Trichomonads x 10'3
+ Growth after 72 hours
* Significantly different from

control (P<0.05)

 

12

media) on each side, allowed the assay of chemoattraction as well as
chemorepulsion. To assay chemokinesis separately from chemotaxis, the
assay was performed eight times with nutrient medium or PBS on both
sides of the filter. The chemotactic agent did not affect
chemokinesis, as membrane trapping was the same, with a <32 difference
whether in PBS or DL8 medium. The chemotaxis of trichomonads towards
nutrients was verified by the increased movement and subsequent
membrane filter trapping of organisms towards nutrient medium and away
from PBS. There were no differences in chemotaxis between trichomonad

species towards or away from nutrient media.

Experiments were performed to find the maximum response by
independently adjusting incubation times and hormone concentrations.
Incubation times ranged from five minutes to 120 minutes and were
found to be maximal at 60 minutes, with no additional difference
compared to controls after 120 minutes. There was a significant
difference in chemotaxis of organisms exposed to estradiol as compared
to controls after only 10 minutes, and the difference was quite linear
up to 60 minutes, at which time it leveled off but did not decrease.
Estradiol concentrations ranged from .001 ng/ml to 1000 ng/ml. The
effect was dose dependent between .01 ng/ml and 100 ng/ml with no
greater effect seen at higher concentrations. All effects could be
blocked with 1 ng/ml tamoxifen; however, tamoxifen alone, at
concentrations up to 100 ng/ml, had no effect on the migrating
trichomonads. All trichomonads not trapped were motile at the end of

experiments (verifiable by light microscopy).

13

Using this assay system, when supplemental estradiol (long/ml) was
placed on top of the membrane filter, 15-402 fewer T. vaginalis were
trapped in the membrane filter than in the control (chemorepulsion).
Similarly, the organisms moved away from their area of containment if
supplemental estradiol was present in the bottom well incubation
medium, and more were trapped in the membrane filter than for control
medium (Table 3). Further, these results are all dose-dependent and
occur with estradiol concentrations beginning at .01 ng/ml. Other
hormones had no effect on T. vaginalis strains. Two isolates of I;
foetus and two of T. gallinae were each found to be totally

unresponsive to estradiol with the same assay (Table 3).

Adherence. Initial experiments were performed to find the maximum
response by varying incubation times and hormone concentrations.
Incubation times were between 15 minutes and 240 minutes and were
found to be maximal at 30-45 minutes. There was a significant
decrease in adherence of organisms exposed to estradiol after 30
minutes when compared to controls. Estradiol concentrations ranged
from .001 ng/ml to 1000 ng/ml. The most significant difference
between controls and estradiol exposed trichomonads was seen with
lng/ml (3.7 x 10'9M). The effect was dose dependent between .01 ng/ml

and 10 ng/ml, with no greater effect seen with higher concentrations.

The adherence of radiolabelled trichomonads to cultured McCoy cells
was maximal after 30-45 minute incubation with a ratio of trichomonads
to mammalian cells of 10:1. Momentary exposure of the trichomonads to

estradiol was not associated with decreased attachment and more

14

Table 3. Effect of 10ng/ml 17-B-Estradiol on the
Chemotaxis of trichomonads#

 

 

 

Chemotaxis+
Top (Estradiol Bottom (Estradiol
Isolate above filter) below filter)
Control (no Estradiol) 13.8 i 0.8 13.8 i 0.8
T. vaginalis-l 9.9 i 1.3* 20.7 i 0.9*
T. vaginalis-Z 9.7 i 1.3* 19.3 i 1.0*
T. vaginalis-3 9.2 i 1.0* 20.8 i 0.9*
T. foetus-1 13.3 i 0.9 13.6 i 0.7
T. foetus DI/#3 14.0 i 0.9 13.6 i 1.1
T. gallinae 30227 13.6 i 1.0 13.8 i 0.7
T. gallinae 30230 13.7 i 1.1 13.6 i 0.9

# All data expressed as mean 1 standard deviation number of
trichomonads x 10'3

+ Trichomonads on filter after 45 minute incubation

* Significantly different from control (P<0.05)

15

prolonged (up to 4 hours) incubation of trichomonads with estradiol
prior to adherence was not associated with further decreases in
attachment compared with 1 hour incubation. Incubation of T;
vaginalis strains with estradiol at lng/ml (3.7 x 10'9M) for 1 hour
prior to incubation with receptor cells was associated with a
statistically significant decrease in adherence to fibroblastic McCoy
cells (Table 4). Again, tamoxifen alone, incubated with T. vaginalis
at concentrations up to lOOng/ml for 1 hour did not alter subsequent
adherence, however, addition of tamoxifen with estradiol in the
incubation medium blocked the effects of estradiol. There were no
statistically significant differences in adherence to McCoy cells with

either T. foetus or T. gallinae after exposure to estradiol (Table 4).

[3H] 17-B-estradiol binding. Protein content of cytosol specimens

were as follows: 75.3 i 7.5 ug/ml from 5.0 x 106 T. vaginalis-l, 85.2

 

: 7.3 for T. foetus-1, and 82.9 i 7.9 for T. gallinae - 30227 (results

 

with other isolates were similar). All isolates displayed specific
estrogen binding. The proteinaceous nature of the [3H] 17-B-estradiol
binding site in trichomonads was assessed by repeating estrogen
binding studies using cytosol incubated with trypsin or PBS control as
described in the Methods section. Specific binding after trypsin
exposure averaged 8.2 i 11.0% of control binding for the 5 trichomonad
isolates tested. The possibility that binding sites in cytosol
preparations were contaminated with residual medium was tested with
radiotracer studies. These studies determined that cytosol

preparations contained less than 0.000022 medium (v/v) and 0.00012

16

Table 4. Effect of 17-B-Estradiol on the Adherence of Trichomonads#

 

 

Adherence+

Isolate Control l7-B-Estradiol (lng/ml)
T. vaginalis-l 536 i 41 156 i 40*

T. vaginalis-Z 549 i 93 213 i 41*

T. vaginalis-3 433 i 70 219 i 52*

T. foetus-1 347 i 94 349 i 83

T. foetus - DI/#3 396 i 92 397 i 90

T. gallinae - 30227 307 i 12 298 i 19

T. gallinae 30230 320 i 39 321 i 33

# All data expressed as mean : standard deviation number of
Trichomonads x 10'3

+ Adherent organisms to McCoy cell monolayer after 45 minute
incubation

* Significantly different from control (P<0.05)

17

(v/v) of either growth or CTLM medium in cytosol buffer yielded no

specific binding of 8 x 10'10 M 3H-estradiol.

Estrogen BindinggAffinitygand Binding7Capacity. Scatchard analysis

 

permits the estimation of the number of receptor sites, i.e., specific
binding by subtracting nonspecific binding from total binding17. This
is done by plotting bound steroid (fmole) on the X axis vs. bound/free
steroid on the Y axis. The values obtained for specific binding can
be used to estimate the number of binding sites and the dissociation
constant (Kd) of the receptor-steroid complex. The Kd is equal to the
concentration of free hormone at which one-half of the receptor sites
are bound. The affinity constant (Ka) is the inverse of Kd or minus
the slope (m), (Ka=-m=1/Kd). The theoretical maximal binding is given

by the intercept of the line joining the points extrapolated to the X
axisl7’18.

The estrogen binding affinity (Ka,Kd) was similar for all isolates;
however, there were significant differences in estrogen binding
capacity comparing T. vaginalis with the T. gallinae and
Tritrichomonas foetus (Table 5). T. vaginalis displayed significantly
greater total estrogen binding capacity compared with the non-estrogen

responsive trichomonads.

Summary and Conclusions
The attachment of microorganisms to cell surfaces is recognized as the
initial step in colonization and subsequent evolution of disease, and

bacterial adherence has therefore been closely studied26. The host

18

Table 5. Estrogen binding of trichomonads#

 

 

Isolate Dissociation constant (Kd)+ Binding capacity
T. vaginalis l 2.9 i 0.9 40.6 i 5.3

T. vaginalis 3 3.2 i 1.3 48.3 i 9.44

T. foetus -1 2.1 i 0.9 3.0 i 0.8j

T. foetus DI #3 2.3 i 1.2 2.9 : 0.9'f

T. gallinae 30227 3.1 i 1.1 11.2 i 6.2?

T. gallinae 30230 3.0 i 1.7 7.3 i 3.4x

data expressed at mean i standard deviation

x10"10 moles/liters [Kd=1/Ka (affinity constant)]

x102 fmol bound/mg cytosol protein

p<0.005 compared with T. vaginalis-1 using one-tailed t-test

+=t

 

19

cell-microorganism interaction involves specific receptors sites on
host cell surfaces and specific ligands on the microorganism527. The
degree of attachment can be determined in vitro by mixing cells and
microorganisms under standard conditions and determining the mean
number of microorganisms adherent per cell. In vitro adherence of
Neisseria gonorrheae, group B Streptococci, E. coli, Proteus mirabilis
and Candida albicans to epithelial cells isolated from the female
genitourinary tract has been observed28'29’30. There have also been
reports that the degree of attachment changed with the stages of the
menstrual cycle of the cell donors and cyclic changes in adherence
were reported for all of these bacteria28’3l. These data support
ideas that hormonal changes may influence bacterial adherence by
altering host epithelial cell number and morphology or by modifying
receptor site synthesis on epithelial cells. It does not consider

direct effects of the hormone on the microorganisms.

In vitro results of hormone acting on trichomonads show that estradiol

directly inhibits the growth and adherence of T. vaginalis to

 

mammalian cells and that estradiol acts as a chemorepellant of I;
vaginalis. This is another example of how hormones may alter
infectivity of microorganisms. It is interesting that trichomonads
have the ability to actively move away from a chemorepellant
(estradiol) after minutes of exposure while the same chemorepellant
takes hours to days longer to effect a demonstratable growth
inhibition upon the organisms. Estradiol does not significantly alter

these parameters in non-human trichomonads parasites.

20

Estrogen binding and estrogen initiation of target cell responses have
been closely studied. For steroid hormones, receptors are found
initially in the soluble intracellular compartment of the cell. After
the steroid binds, the receptors modified by the bound hormone, attach
to the chromatin of the target tissue cell, and are then found
predominantly in the nucleus. Estrogens arealipophilicand therefore
may penetrate cell membranes simply by passive diffusion, although
specific uptake mechanisms have been postulated32. Binding of the
hormone by the receptor occurs rapidly inside the cell. This
interaction in turn results in a change in the hormone-receptor
complex so that the complex then binds to the nuclear chromatin. The
binding of receptor-steroid complexes with the nuclear chromatin
results in changes in the levels of specific mRNAs. Interestingly,
all species of trichomonads retain estrogen binding sites of similar
affinity, but T. foetus and T. gallinae, which are not primary
pathogens of females have significantly less estrogen binding sites.
T. vaginalis isolates, which display physiologic responsiveness to
estrogens, have significantly higher estrogen binding capacity than
the non-human trichomonads, which do not display physiologic estradiol
responsiveness. The binding affinity was similar for all isolates and
was quite high. Steroid receptors are expected to have a high
affinity for their respective hormones. This is anticipated because
the mammalian (or human) blood levels of steroids are usually 10‘10M -
10'8M. If a tissue or organism within a host is to respond to a
hormone via a receptor mechanism in which the hormone binds to the
receptor, then the receptor must have an affinity for the hormone

which is in the physiologic range25’33.

Unfortunately, effects of estrogens on potentially infecting
microorganisms may be very different from the effects on the host. It
seems that estrogens decrease the infectivity of T. vaginalis lg
yitgg, but the few studies available suggest that estrogens increase
infectivity of T. vaginalis in viv022’34’35. Thus, interplay between
potentially divergent effects of hormones on microorganisms and the
host ultimately determines alterations in infectivity. However, the
presence of specific estrogen receptors in T. vaginalis may allow the
use of hormonal and antihormonal therapy in the treatment of

infections by these organisms.

10.

11.

12.

22

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