Adult BALB/c mice of both sexes were infected intranasally with 106 viable P. brasiliensis conidia. Animals were sacrificed at intervals up to 6 months and studied by histopathology and organ cultures. At the time of challenge lung sections showed that instilled conidia had reached the alveoli; at 12 h such conidia were transforming into yeast cells, with multiple buds appearing by 18 h. Initially, the cellular infiltrate was composed of polymorphonuclear leukocytes; 6 days later, lymphocytes, plasmocytes and macrophages predominated. Multinucleated giant cells appeared only after 6 weeks. The rate of pulmonary infection as determined by organ culture was high (82 of the 83 mice studied). The experimental infection was progressive as indicated by increasing numbers of viable fungi with time. The results of this study demonstrate that the conidia produced by P. brasiliensis mycelial form are infectious, producing active disease in healthy animals.

Evidence that disease due to the thermally dimorphic fungus Paracoccidioides brasiliensis occurs postpuberty predominantly in males led us to hypothesize that hormonal factors critically affect its pathogenesis. We show here that estrogens inhibit mycelialto yeast-form transformation of P. brasiliensis in vitro. Transformation of three isolates was inhibited to 71, 33, and 19% of the control values in the presence of 10-10, 10-8, and 10-6 M 17i-estradiol, respectively. The synthetic estrogen diethylstilbestrol was active but less potent than estradiol, whereas testosterone, 17a-estradiol, tamoxifen, and corticosterone were inactive. This function was specifically inhibited, since yeast-to-mycelium transformation, yeast growth, and yeast reproduction by budding were unaffected by 171-estradiol. Of note is the fact that mycelium-to-yeast transformation occurs as the first step in vivo in the establishment of infection. The cytosol of the three isolates studied possesses a steroid-binding protein which has high affinity for 17j3-estradiol. We believe that this binding protein represents a P. brasiliensis hormone receptor which can also recognize mammalian estrogens. We hypothesize that the ability of estrogen to decrease or delay mycelium-to-yeast transformation at the initial site of infection contributes to or is responsible for the marked resistance of females, and that the binder described is the molecular site of action.

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Conidia produced by Paracoccidioides brasiliensis are inhibited by mammalian estrogens in their in vitro conversion into yeast-form cells. This was demonstrated with four different isolates. In these experiments, conversion was reduced to 10.7 and 34.4% of the control values by 17-beta-estradiol at 10-6 and l0-8 M, respectively. At the same concentrations, the synthetic estrogen diethylstilbestrol was slightly less inhibitory. In contrast, other sex hormones and analogs, i.e., testosterone, 17-alpha-estradiol, tamoxifen, and hydroxytamoxifen, had no effect on conidium-to-yeast conversion. Previous studies have shown that estrogens similarly inhibit mycelium-to-yeast-form transition in P. brasilensis. Conidia, and not mycelial fragments, are believed to be the natural infectious propagules. These findings with conidia support the hypothesis that estrogens, affecting the initial host-parasite interactions by suppressing conversion to the parasitic form of the organism, are, at least in part, responsible for the greater resistance of females to paracoccidioidomycosis.

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The ultrastructure of asexual spores (conidia) produced by the mycelial form of Paracoccidioides brasiliensis was studied for the first time with transmission electron microscopy, using thin sections of aldehyde-osmium-fixed and epoxy-resin-embedded samples. The various types of conidia observed in the sections correlated well with previous light-microscopic descriptions. These types were intercalary or apical conidia, depending on their location along the originating hyphae. As in previous studies they were characterized as arthroconidia, aleuriospores and sessile or pedunculate pyriform conidia. The sporogenous cells were clearly distinguished from hyphal cells by the thickness and appearance of their cell walls. Copious fibrillar material (glycocalyx) detected at the cell surface was stained with ruthenium red during the fixation process. Typical subcellular organelles (nucleus, nucleolus, mitochondria, ribosomes, etc) were found in most of the sections. It was concluded that the spores produced by the mycelial phase of P. brasiliensis possess all attributes of viable and physiologically competent eukaryotic cells.