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Infection and Immunity, November 1998, p. 5587-5591, Vol. 66, No. 11
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Morphological Transition of Paracoccidioides
brasiliensis Conidia to Yeast Cells: In Vivo Inhibition in Females
Beatriz H.
Aristizabal,1
Karl V.
Clemons,2,3
David A.
Stevens,2,3,* and
Angela
Restrepo1
Corporacion para Investigaciones Biologicas,
Medellin, Colombia1;
Division of
Infectious Diseases, Department of Medicine, Santa Clara Valley Medical
Center and California Institute for Medical Research, San Jose,
California 951282; and
Division of
Infectious Diseases and Geographic Medicine, Department of
Medicine, Stanford University Medical School, Stanford, California
943053
Received 27 October 1997/Returned for modification 18 December
1997/Accepted 14 August 1998
 |
ABSTRACT |
Clinical paracoccidioidomycosis is 13 times more common in men than
in women. Estrogen inhibits the transition of mycelia or conidia (the
saprophytic form of Paracoccidoides brasiliensis) to yeasts
(the parasitic form) in vitro. Here, we show that, in male mice that
were infected intranasally (mimicking natural infection) the transition
of conidia in bronchoalveolar lavage fluids to intermediate forms and
yeasts occurred over 24 to 96 h; CFU and yeasts (shown by
histopathology) increased subsequently. In females, transition did not
occur and infection cleared. These events in vivo are consistent with
epidemiological and in vitro observations, suggesting that female
hormones block transition and are responsible for resistance.
| |
TEXT |
Paracoccidioidomycosis is one of the
most important systemic mycoses affecting residents of Latin America.
Its etiological agent is the dimorphic fungus Paracoccidioides
brasiliensis (2). This organism probably occurs in the
mycelial form (M) in nature, and the disease is thought to be acquired
by inhalation of the propagules produced by this phase. Clinical
manifestations are reflected in diverse forms, ranging from
asymptomatic pulmonary lesions to systemic generalized infections. The
tissue form of P. brasiliensis is a multiply budding yeast
(Y) (26, 33).
Clinical disease is more common in adult males, with a male/female
ratio of 13:1 in some areas where it is endemic (2, 20, 33).
In Colombia this ratio is 78:1 (20). The greater incidence
of clinical disease in adult males led to the hypothesis that hormonal
factors might play a role in the pathogenesis of paracoccidioidomycosis
(15, 23, 30). In contrast to overt disease, subclinical
infection, which is detected by delayed-type hypersensitivity to
paracoccidioidin in healthy individuals from areas where the disease is
endemic, does not reveal this sex-based difference. Thus, it appears
that both sexes acquire subclinical infections at the same rate
(19) but that progression to overt disease is much more
frequent in males (33).
A number of hormonal systems in various fungi have been described
(10, 30). Several fungi have cytosolic proteins that bind
mammalian hormones with high affinity and stereospecificity (8,
15, 30, 31).
Data from in vitro studies also support the hypothesis that hormones
might influence the pathogenesis of paracoccidioidomycosis in humans.
In vitro, the inhibition of transition of conidia (C) or M to Y by
estrogens has been described (23, 25). However, the in vivo
effect of the female hormonal milieu on the transition of C to Y has
not been described previously.
The primary objective of this study was to examine the possible in vivo
effect of the different hormonal environments in the different sexes on
the pathogenesis of the experimental infection induced by C in BALB/c
mice.
Fungus and inoculum.
P. brasiliensis ATCC 60855 (21) in M was used to produce C (22). The C
viability was determined by ethidium bromide fluorescence (3), and the inoculum was adjusted so that 3 × 106 to 4 × 106 viable propagules were
contained in 0.06 ml. The inoculum was administered by intranasal
instillation under methoxyflurane anesthesia (24).
Animals.
Specific-pathogen-free male and female BALB/c mice
from Simonsen Laboratories (Gilroy, Calif.) were used. The animals were 4 to 6 weeks old and were supplied with sterilized food and acidified water ad libitum.
Three mice of each sex (of a total of 33 males and 33 females) were
euthanized per interval postinfection (1, 24, 48, 72, and 96 h) for bronchoalveolar lavage (BAL) and for CFU determination and
histopathology at 2, 4, and 6 weeks. The results of BAL studies were
confirmed in a second identical experiment of the same proportions.
BAL and lung examination for fungal morphology and
quantitation.
Samples were collected after the mice were
euthanized with CO2. The lungs were inflated with RPMI 1640 medium; 0.5-ml volumes were injected and withdrawn several times to
obtain samples of BAL fluid. This suspension was centrifuged at 4°C,
and the pellet was suspended in 2 ml of physiologic saline. After
harvesting of the cells, cytospin slides were prepared and stained with
Grocott methenamine-silver nitrate stain (GMS). Fungal cells were
counted, and their morphology was assessed (C, intermediate [INT], or
Y). The number of fungal cells was expressed as the log10.
Because of the rarity of fungal cells in BAL fluids
96 hours
postinfection in preliminary experiments, we monitored the course
of
the infection at later times by quantitating CFU and examining
the
pulmonary histopathology. Lungs were removed, weighed, and
homogenized
in tissue grinders. The homogenates were serially
diluted in saline and
plated in duplicate on modified Sabouraud
glucose agar plates with
0.01% thiamine (Mycosel; BBL, Cockeysville,
Md.). The plates were
incubated at room temperature for 2 months.
Mean colony counts were
obtained and expressed as log
10 CFU per
entire organ. For
histopathology, lungs were removed and fixed
in 10% buffered formalin
and were paraffin embedded. Serial sections
were stained with GMS. The
fungi were counted, and their morphology
was assessed.
Hematoxylin-and-eosin-stained sections were used
to observe the
inflammatory response and the tissue cellularity.
Statistical analyses.
Statistical analyses were done on the
pooled data from two experiments for the total fungal cells recovered
from BAL fluids and for the proportions of C, INT, or Y from the sexes
at various times. Analyses of the total fungal cells recovered at each
time were done with a Mann-Whitney U test on log10
transformed numbers (GB-STAT, version 6.0; Dynamic Microsystems, Silver
Spring, Md.). Changes with time were analyzed with a Kruskal-Wallis
one-way analysis of variance (ANOVA). Comparative analyses of the
proportions were done by parametric tests with a one- or a two-way
ANOVA. Proportions were first transformed with an arcsin transformation to normalize the data (29). An analysis of the changes over time for each sex was done with a one-way ANOVA. Comparisons between sexes with time were done with a two-way ANOVA, and comparisons at each
time between sexes were done with a one-way ANOVA, with the C data
considered alone and the INT and Y data grouped together. Analyses of
the CFU and fungal cell counts in the histologic preparations were done
with Student's t test. A P value of <0.05 was
considered significant.
Fungi in BAL fluid.
At 1 h postinfection there was no
difference between sexes in the morphology of the fungus, because only
C were observed (Fig. 1). In the males,
INT and Y were observed as early as 24 h postinfection. In
contrast, few INT and Y were observed in BAL fluids from females up to
96 h.
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FIG. 1.
Appearance of P. brasiliensis cells at 1, 24, 48, 72, and 96 h, in the BAL fluids of mice infected intranasally
with C. Data are pooled results from two experiments (n = 6, except for females at 96 h, n = 5) and
show total fungal cells (A) and percentages of total cells as C (B),
INT (C), and Y (D). C. I., confidence interval; SD, standard
deviation.
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|
The total fungal cells recovered in BAL fluids significantly diminished
with time in males and females (
P values of <0.0001
and
<0.0009, respectively) (Fig.
1A). A significantly greater
number of
fungal cells was recovered from males at 24, 72, and
96 h
(
P values of <0.002, <0.002, and <0.004, respectively).
Two major factors affected the morphological transition of the
organisms: gender and the length of time after infection. Analyses
of
the proportions of the C, INT, and Y forms showed that both
factors
were significant in their effects on morphological transition
(
P < 0.0001) (Fig.
1B to D). In males the proportion
of fungal
cells remaining as C significantly decreased with time
(
P < 0.0001),
while numbers of INT and Y increased
(
P < 0.0001).
Comparisons between genders of the proportions of fungal cells
appearing as C, INT, and Y at different times showed significant
differences at 24 to 96 h. The proportion of C at 24 to 96 h
was
significantly higher for females (
P < 0.004, at
all times), and
the proportion of cells as INT or Y was significantly
greater
at 24 to 96 h in males (
P < 0.004, at all
times).
The various stages in the C-to-Y transformation are illustrated in Fig.
2. The inoculated propagules (Fig.
2A)
changed over
the observation time in males. Initially, C converted into
INT
(Fig.
2B) and later to Y. Multiply budding Y (Fig.
2C) were
observed
only toward the end of the 96-h period.
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FIG. 2.
Stages in the C-to-Y transition in lungs of mice. (A)
Inoculum of C obtained from M phase in BAL fluid 1 h
postchallenge, stained with GMS. Magnification, ×40. (B) INT cells
developing in males 48 h postchallenge, stained with GMS.
Magnification, ×100. (C) Y cells in males 48 to 96 h
postchallenge, stained with GMS. Magnification, ×100. Note that the
diameter of Y (cells in the plane of focus) is twice that of cells in
panel B, and note the reproduction by budding in Y.
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|
CFU in lung cultures.
Infected males were unable to control
the infection, as shown by CFU recovered from the lungs at 2 to 6 weeks
(Table 1).
Histopathology of the lungs.
In the experiments enumerating
fungal cells in lung sections at 2, 4, and 6 weeks postchallenge,
significant differences were observed between male and female mice. In
females, fungal cells were not found at any time. In contrast, in
males, 10 to 20 fully transformed Y were counted per section
(P < 0.05).
Histological observation showed a chronic granulomatous inflammatory
response in males. Lesions were localized in the peribronchiolar
and
perivascular spaces (Table
2) with many
histiocytes and lymphocytes
at all times; as the lesions evolved, some
plasmacytes and giant
cells appeared. In contrast, no granulomas,
plasmacytes, or giant
cells were seen in females, and histiocytes were
less numerous
and later disappeared (Fig.
3).
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FIG. 3.
Histological observation of the lungs of mice 2 to 6 weeks after intranasal infection with P. brasiliensis C. (A)
Chronic granulomatous reaction in a male mouse (hematoxylin and eosin
stain; magnification, ×40). (B) Lung section with no inflammatory
reaction in a female mouse (hematoxylin and eosin stain; magnification,
×40).
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Various observations suggest sex-based differences in the immune
response (
6,
9,
11,
18,
32). Gender-associated
differences
in patterns of resistance to bacterial, viral, or
parasitic infection,
tumor development, and autoimmune responses
are well documented
(
6,
13,
17,
32). Women appear to
be less susceptible to some
fungal, bacterial, and viral infections,
presumably due to hormonal
factors (
7,
32). It has been reported
that natural
estrogens, such as estradiol, strongly stimulate
macrophage activity,
whereas testosterone has only a marginal
effect (
9,
11,
32).
Active and progressive paracoccidioidomycosis is seen markedly more
frequently in men (
33). This suggests that female sex
hormones are important in the progression of subclinical
paracoccidioidal
infection towards overt disease and that small
differences in
hormonal influence on immune responses (as cited above)
are greatly
augmented by a hormone-specific effect on this pathogen. In
vitro
the direct inhibitory effect of estradiol on the M- or C-to-Y
transformation has been shown (
23,
25). These observations
and the finding of estrogen-binding proteins in the cytosol of
P. brasiliensis suggest that endogenous host estrogens may inhibit
the adaptation of the fungus to tissue conditions (
15,
31).
Animal experimentation has contributed little to our knowledge
concerning the influence of sex hormones in paracoccidioidomycosis
or
the hormonal influence on
P. brasiliensis adaptation to host
conditions (
30). When infected experimentally with Y inocula
by the intratracheal route (
4,
5) or by the intraperitoneal
route (
1), female mice were more resistant than males. In
contrast,
in intraperitoneal infection (
12), intact
female rats were more
susceptible to infection with Y. McEwen et al. (
16), using a
C inoculum given
intranasally, demonstrated that severe pulmonary
paracoccidioidomycosis
occurred at the same rate in female and
male mice. The susceptibility
of female mice during the various
stages of the estrous cycle to
intravenous inoculation with Y
of
P. brasiliensis has been
compared with that of males (
27,
28). Female mice,
especially those at metestrus II, developed
invasive disease more often
(
27,
28). Generally, at proestrus
and estrus, the blood
estrogen is at its highest level, while
at metestrus II, it is at its
lowest (
14,
28). These experiments
showed that female
hormones affect the clearance of
P. brasiliensis Y from
tissues.
However, five of the six experiments mentioned (
1,
4,
5,
12,
27,
28) used Y inocula, i.e., the tissue form
of the fungus. The use
of Y circumvents natural events because
the infectious particles (M
fragments or C) are presumably M derived.
C were used in only one study
(
16). There were differences with
our study: the use of mice
that were 12 weeks old (
16); concentration
of the inoculum
(fourfold lower) (
16); the anesthetic used,
diethyl ether
(
16), known to destroy alveolar lining, versus
methoxyflurane in our study, the source of BALB/c mice (Corporacion
para Investigaciones Biologicas) (
16); and the experiment
duration
(up to 6 months) (
16). On the other hand, the
P. brasiliensis strain used was the same, identified as Gr
(
16); later, the
strain was registered in the American Type
Culture Collection
as ATCC 60855. In additional experiments
(
32a), it was also found
that female BALB/c mice, 6 to 8 weeks old, were resistant to intravenous
challenge with C. In those
experiments, the number of CFU in lungs
and spleen were significantly
lower in females 3 weeks after challenge.
The infection continued to
progress in males 4 to 8 weeks after
challenge, whereas in females the
infection had cleared by 4 weeks.
In contrast, intravenous infection
with Y was cleared from lungs
and spleens in both sexes at the same
rate.
To prove that sex hormones did indeed have an inhibitory effect in the
transition of
P. brasiliensis from C (infectious form)
to Y
(parasitic form), we studied sequential stages of the transition
in
mice. In males, even during the earliest stages of the infectious
process (24 to 48 h), C had already begun to transform, with many
INT cells and even some Y. This indicates that, at least in males,
the
transition of C to Y cells does not take long. Other studies
have shown
that after 96 h, a large proportion of C had already
transformed
into Y (
16). Remarkably, in the present study, during
the
same interval, C but no INT or Y cells were found in females.
We noted during examination of the BAL preparations that although
macrophages comprised
95% of host cells in both sexes prior
to
infection, polymorphonuclear neutrophils comprised the majority
of
cells in males after infection, whereas in females, cells other
than
macrophages were rarely seen. This suggests that the chemotactic
response might be hormonally modulated. Thus, a hormonally mediated
influence on the immune system could be an additional or adjunct
explanation for the observed sex-based differences in resistance.
The
different profile could also relate to different chemotaxins
and
chemotaxinogens produced by different morphological forms
of the
fungus, i.e., INT and Y eliciting polymorphonuclear neutrophils
in
males and C eliciting macrophages in females. In addition,
the
macrophage response may be the more protective one (
11,
13).
These findings suggest that the inhibition of the fungal transition
process by female hormones results in protection. In addition,
it is
possible that males suffer a suppression of their cell-mediated
immune
responses to
P. brasiliensis due to androgens, as androgens
have been shown to possess both immunoinhibitory and immunostimulatory
capacities (
13,
17).
The histopathology samples obtained at later time points showed the
presence of abundant budding Y, accompanied by an intense
inflammatory
response with epithelioid granuloma formation. Also,
numbers of CFU
from lungs were higher in males. This suggests
that males develop
chronic and progressive paracoccidioidomycosis
more frequently, because
females are able to halt the transformation
process before 96 h.
The female immune response was strong enough
to kill the fungus, as
indicated by negative cultures
2 weeks
postchallenge.
The initial events that we describe demonstrate, for the first time in
vivo, that female resistance to paracoccidioidomycosis
is related to
early events after infection and that these events
might be hormonally
modulated. These findings support our previous
hypothesis, derived from
in vitro observations (
23,
25), that
female hormones inhibit
the transition of
P. brasiliensis from
M or C to Y, thus
making females less susceptible to paracoccidioidomycosis.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the Instituto Colombiano para el
Desarrollo de la Ciencia y la Tecnologia Francisco Jose de Caldas
(COLCIENIAS), Bogota, Colombia, and the Corporacion para
Investigaciones Biologicas, Medellin, Colombia.
We thank B. L. Gomez for technical assistance, R. Azzi for
assistance with histopathology, E. Brummer for contributions to BAL
data, and L. Treat-Clemons and F. Montoya for the statistical analyses.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, 751 S. Bascom Ave., San Jose, CA 95128-2699. Phone: (408) 885-4313. Fax: (408) 885-4306. E-mail:
stevens{at}leland.stanford.edu.
Editor:
T. R. Kozel
| |
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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