Paracoccidioides brasiliensis

P. brasiliensis is a thermally dimorphic fungus, which causes the most prevalent systemic mycosis (paracoccidioidomycosis) in Latin America. The fungus exists in soil as a filamentous form, which transforms into a yeast form when it invades mammalian hosts or by a temperature switch from room temperature to 37°C, in vitro. Infection is initiated after inhalation of conidia or mycelial fragments by the mammalian host. These mycelial propagules further differentiate into the yeast form of the organism, which is found in the tissues. The transition of mycelial to yeast form is crucial for the establishment of disease. Epidemiologically, clinical disease is more common in adult men than women despite equal frequencies of exposure to this fungus, and it has been estimated that females are about 13-70 times less likely as males to develop clinical disease (Brummer et al. 1993). Furthermore, the development of clinical disease is equal in males and prepubertal or postmenopausal females. Thus, this led to the speculation that human sex hormones might have an effect on progression of this disease.

Using a microculture system, morphological transformation of mycelia to yeast form of clinical isolates of P. brasiliensis was tested in the presence of various steroids, including 17b-estradiol (E2), testosterone, tamoxifen and 17a-estradiol, and the nonsteroidal estrogen, diethylstilbesterol (DES), at concentrations ranging from 2 x 10-10 to 2 x 10-6 M to span relevant physiological and pharmacological concentrations (Restrepo et al. 1984). Mycelial to yeast transformation was dose-dependently inhibited by E2 to 71% (2 x 10-10), 33% (2 x 10-8) and 19% (2 x 10-6 M) of the transformation of control cultures. In addition, DES at these concentrations resulted in 85% (2 x 10-10), 54% (2 x 10-8) and 37% (2 x 10-6 M) inhibition when compared to control. Testosterone, tamoxifen, and 17a-estradiol were inactive. Inhibition of the morphological transformation occurs only in one direction and none of the tested compounds blocked the transformation of yeast to mycelia nor did they affect yeast growth or budding. Similarly, the transformation of conidia to yeast was shown to be blocked by E2 (Salazar et al. 1988). Thus, inhibition of transformation of conidia or mycelia fragments to yeast form is a biologically specific phenomenon with E2 effective and its stereoisomer 17a-estradiol ineffective.

To identify a possible steroid hormone receptor in the cytosol of P. brasiliensis, yeast cells were grown and disrupted with glass beads to produce a cytosolic extract. Specific binding activity was examined by addition of [3H]-E2 and 500-fold molar excess of unlabeled E2 to the cytosol to perform conventional steroid binding assays (Loose et al. 1983b). Bound hormone was separated from free hormone using centrifuged Sephadex microcolumns. In a single point study using 130 nM [3H]-E2 specific E2 binding was demonstrated in the range of 200 fmol/mg of cytosol protein. Some specific binding was detected with [3H]-progesterone (60 fmol/ mg protein) and DES (42 fmol/mg protein); only trace binding was detected with dihydrotestosterone and testosterone, and no binding was seen with corticosterone. Progesterone binding was completely blocked by E2, suggesting the presence of a single estrogen binding site with some cross reactivity for other steroids. However, the presence of a separate progesterone or DES binding site has not been completely excluded.

More extensive binding studies with cytosol and [3H]-E2 done at 0°C indicated that binding was maximal after about 90 min of incubation (Loose et al. 1983b). The dissociation rate was determined and the off-rate time for half of the bound hormone to be released was 29 min. Binding characteristics at equilibrium showed binding to be saturable with low nonspecific binding. Scatchard plots suggested a single class of noninteracting binding sites, with a dissociation constant (Kd) of 8.5 nM and a binding capacity (N^) of 210 fmol/mg protein, indicative of high-affinity low capacity binding. The specificity of the binding was assessed in competition assays, where unlabelled hormones compete for specific binding with [3H]-E2. Competition for bound E2 was maximal with E2, whereas the related estrogens, estrone, and estriol, had only about 25% of the affinity of E2. DES was a weak competitor, in contrast to its affinity for the mammalian E2 receptor. Thermal stability studies at 0, 37, and 56°C for over 30 min showed a second binding protein, with lower affinity at 37°C. However at 56°C, only the high capacity (Nmax 1700-2600 fmol/mg protein) binding protein was detected. Treatments with DNase, RNase, and phospholipase A2 had little effect on the [3H]-E2 binding, whereas binding activity was inhibited by trypsin and reduced by N-ethylmaleimide. These results together suggested specific binding was due to a protein containing sulfhydryl groups. Liquid chromatographic (HPLC) studies indicated that the binding protein has a relative molecular mass of 60,000 Da and sucrose gradient centrifugation indicated a sedimentation coefficient of 4.4S.

Because the functional response of inhibition of form transformation was that of inhibiting the mycelial form, it was important to determine whether the mycelial form also contained a specific binding protein for E2, and we were able to devise ways to work with the mycelial form of P. brasiliensis for binding assays (Stover et al. 1986). A binding protein was discovered in this form, in many isolates of the fungus, as had been shown for the yeast form. The Kd and Nmax were 13 nM and 78 fmol/mg protein, respectively. In addition, a second low-affinity (Kd 150 nM) high-capacity (Nmax 3,000-4,500 fmol/mg protein) binding protein was demonstrated in the yeast form. In competition studies, DES was a potent competitor for E2 with the mycelial binder, correlating better with its functional effect of inhibiting the mycelium-to-yeast transformation described earlier.

To examine the cellular response of P. brasiliensis to the presence of E2, we followed temporal protein expression during mycelial to yeast transformation using [35S]-methionine incorporation and 1-D SDS-PAGE (Clemons et al. 1989a). E2 altered protein expression in P. brasiliensis in vitro when shifted to the temperature that permits mycelium-to-yeast transformation, blocking the appearance of proteins associated with transformation or yeast and maintaining a more mycelial protein profile until later time points, where de novo protein expression was virtually shut down (Clemons et al. 1989a). In addition, exposure of mycelial cultures to E2 without a switch in temperature induced the uptake of labeled methionine (see Figs. 15.1 and 15.2).

In light of the demonstration of specific E2 binding and effects on morphological form transition and protein synthesis, we performed in vivo studies in a murine model of pulmonary infection to determine whether E2 could alter the pathogenesis of P. brasiliensis and explain the observed epidemiologic data of resistance of females. In the initial studies we followed morphologic transformation of conidia

Fig. 15.1 Comparison of proteins from the cytosol fractions of E2-treated P. brasiliensis undergoing M to Y transition with M and Y controls. Proteins were resolved through 9% SDS-PAGE and silver stained. Lane 1, M-cells incubated solely at 25°C, treated with E2 for 24 h; lanes 2, 3, and 4, M-cells after 24, 72, and 120 h of E2 treatment at 37°C, respectively. Lane 5, Y-cells incubated at 37°C only. Lanes 1-4 represent M cultures treated with E2 (2.6 x 10-7 M), and lane 5 represents the Y control. Note the maintenance of the M-form profile at 24 and 72 h (lanes 2 and 3) and the decreased total number of bands by 120 h (lane 4), which demonstrates little similarity to the Y-form profile (lane 5). For reference, the molecular mass in kDa of the transition bands (between lanes 3 and 4) as well as selected M-specific (near left) and Y-specific (near right) bands are indicated. Molecular masses in kDa of standards are indicated on the far left. Reprinted with permission from the Society for General Microbiology, United Kingdom from reference (Clemons et al. 1989a)

Fig. 15.1 Comparison of proteins from the cytosol fractions of E2-treated P. brasiliensis undergoing M to Y transition with M and Y controls. Proteins were resolved through 9% SDS-PAGE and silver stained. Lane 1, M-cells incubated solely at 25°C, treated with E2 for 24 h; lanes 2, 3, and 4, M-cells after 24, 72, and 120 h of E2 treatment at 37°C, respectively. Lane 5, Y-cells incubated at 37°C only. Lanes 1-4 represent M cultures treated with E2 (2.6 x 10-7 M), and lane 5 represents the Y control. Note the maintenance of the M-form profile at 24 and 72 h (lanes 2 and 3) and the decreased total number of bands by 120 h (lane 4), which demonstrates little similarity to the Y-form profile (lane 5). For reference, the molecular mass in kDa of the transition bands (between lanes 3 and 4) as well as selected M-specific (near left) and Y-specific (near right) bands are indicated. Molecular masses in kDa of standards are indicated on the far left. Reprinted with permission from the Society for General Microbiology, United Kingdom from reference (Clemons et al. 1989a)

after pulmonary instillation. Conidial transformation into yeast was not impeded in male mice, with budding yeast forms present as early as 48 h. In contrast, female mice had progressively fewer cells in the bronchoalveolar lavage fluid and no

Fig. 15.2 Comparison of [35S]methionine-labeled proteins in cytosol fractions of E2-treated and untreated P. brasiliensis undergoing M to Y transition. Labeling was done for 2 h in the absence of radioinert methionine prior to disruption of organisms and extraction of cellular proteins. Lanes were loaded with equal counts of [35S]-labeled proteins, electrophoresed and processed for fluorography. Lane assignments: lane 1 , M control; 2, M E2,-treated; 3 and 5, M controls grown at 37°C for 24 h, and 72 h, respectively. Lanes 4 and 6 are M treated with E2 (2.6 x 10-7 M) grown at 37°C for 24 h, and 72 h, respectively. Note the effect of E2 on label incorporation of M-form (lane 2) as compared to untreated control (lane 1). Absence of the 92 kDa Y-specific band in lane 6 is indicated. The fluorogram was intentionally over-exposed to enhance the bands in lane 2. Molecular masses in kDa of standards are indicated on the left. Reprinted with permission from the Society for General Microbiology, United Kingdom from reference (Clemons et al. 1989a)

budding yeasts were observed (Aristizabal et al. 1998). In addition, no CFU were recovered from the lungs of female mice after 2-6 weeks of infection, whereas log10 2 or more CFU were recovered from male mice (Aristizabal et al. 1998). Thus, in female mice, transformation of the conidia was severely impeded, with the mice able to clear detectable infection (see Fig. 15.3). Furthermore, we found that castrated male mice reconstituted with high-doses of E2 initially inhibited conidial transformation to yeast and subsequent proliferation. However, these mice had recurrence of disease with progression later in infection, which we speculate may have been due to the immunoregulatory effects of the high-doses of E2 administered to those animals. In contrast, castrated females reconstituted with testosterone were unable to restrict disease (Aristizabal et al. 2002). Taken together, these studies support the hypothesis that the interaction of the organism with E2 contributes to the resistance of females to this infection.

Fig. 15.3 Histopathology of the lungs in normal mice 4 weeks after infection with conidia of P. brasiliensis (H&E, X100). (a) Normal males (NM) showing an intense chronic inflammatory reaction, also with granuloma formation and presence of yeast cells. (b) Normal females (NF) showing a slight inflammatory reaction, with no yeast cells present. Reprinted with permission of the publisher (Taylor & Francis Ltd, http://www.tandf.co.uk/journals) from reference (Aristizabal et al. 2002)

Fig. 15.3 Histopathology of the lungs in normal mice 4 weeks after infection with conidia of P. brasiliensis (H&E, X100). (a) Normal males (NM) showing an intense chronic inflammatory reaction, also with granuloma formation and presence of yeast cells. (b) Normal females (NF) showing a slight inflammatory reaction, with no yeast cells present. Reprinted with permission of the publisher (Taylor & Francis Ltd, http://www.tandf.co.uk/journals) from reference (Aristizabal et al. 2002)

15.5.2 C. albicans

C. albicans is an opportunistic dimorphic fungal pathogen of medical importance. Studies by Loose et al. (Loose and Feldman 1982; Loose et al. 1981, 1983a), into the evolution of hormone-receptors, represents a classical example of mammalian steroid hormone interaction with the fungus. In those studies, C. albicans was shown to have a protein capable of specific corticosteroid-binding (CBP) that exhibits high affinity for corticosterone and progesterone. Specific corticosterone binding was found in the cytosol of C. albicans with a Kd 6.3 nM and binding capacity Nmax 650 fmol/mg of protein. Specific binding was demonstrated to be due to a protein with apparent molecular mass of 43 kD (Loose and Feldman 1982; Loose et al. 1981). Interestingly, lipid extracts of the cell pellet or of culture filtrate displaced specific [3H]-corticosterone from CBP, and are presumed to contain an endogenous ligand (Loose et al. 1981). Thus, CBP exhibits properties consistent with a receptor molecule. It is stereo-specific, extremely selective with affinity for corticosterone (~7 nM) that is equivalent to that of mammalian glucocorticoid receptors (Loose et al. 1981).

CBP has been found in both serotypes A and B of C. albicans and a survey of various species of Candida indicated that the binding protein appears ubiquitous within the genus (Loose et al. 1983a) and was confirmed in other studies (Powell and Drutz 1983). Although the binding parameters differed between species, Scatchard plots were linear, indicating a single class of binding sites in each. It was shown that corticosterone can enter an intact yeast cell and bind to the protein. Candida growth, yeast to mycelia conversion of Candida or glucose oxidation was not affected by the addition of different steroids (corticosterone, progesterone and dexamethasone) over a range of 1 x 10-6 to 2 x 10-10 M (Loose et al. 1983a).

To determine the relationship of CBP from C. albicans to the mammalian hormone receptors, the CBP gene has been cloned and expressed. It revealed an open reading frame of 1,467 bp that encodes a protein with a molecular weight of 44,545 Da; the expressed protein has the properties of the native CBP. Sequence comparison of CBP gene to members of mammalian steroid-thyroid-retinoic acid receptor gene superfamily showed that CBP is unrelated to these hormone receptors (Malloy et al. 1993). Interestingly, in S. cerevisiae, the FMS1 (fenpropimorph multicopy suppressor gene 1) yeast gene shows a protein identity of 35% with C. albicans CBP (Joets et al. 1996).

C. albicans has also been shown to have an estrogen-binding protein (EBP) that displays high affinity for estradiol and estrone (Powell et al. 1984; Skowronski and Feldman 1989). Specific binding was found for E2 with a Kd of about 6 x 10-8 M and Nmax of 400-13,000 fmol/mg of protein depending on the study and the strain of C. albicans tested (Powell et al. 1984; Skowronski and Feldman 1989). Furthermore, the abundance of EBP was found to be significantly higher in early log phase growth (Skowronski and Feldman 1989). Binding was saturable and other estrogens, estrone and estriol, were the best competitors and unlike the situation of the human estrogen receptor, tamoxifen does not bind to EBP in C. albicans (Powell et al. 1984; Skowronski and Feldman 1989).

Cloning of the EBP gene revealed an open reading frame of 1,221 bp that encodes a protein with 407 amino acids and having a molecular mass of 46,073 Da, the estimated size of EBP (Madani et al. 1994). The expressed gene showed high affinity with binding for estradiol and a competitive profile comparable to wildtype C. albicans EBP. Sequence comparison showed that EBP shares a 46% amino acid identity with the old yellow enzyme, an oxidoreductase from S. cerevisiae, but, as anticipated, was unrelated to the human estrogen receptor. Expressed protein exhibited oxidoreductase activity and showed inhibition by the treatment of E2 in vitro (Madani et al. 1994).

C. albicans has also been shown to interact with the human peptide hormones luteinizing hormone (LH) and human chorionic gonadotropin (Bramley et al. 1990, 1991). Both low and high affinity binding sites have been found for these peptides, and the binding activity has been demonstrated in microsomes and cytosol preparations. Furthermore, LH was found to stimulate germ tube formation (Kinsman et al. 1988), and adenylate cyclase activity (Williams et al. 1990). In addition, other investigators have reported a human chorionic gonadotropin-like protein in extracts of C. albicans that was a potent stimulator of germ tube formation in the presence of serum (Caticha et al. 1993), which is suggestive that this protein is an endogenous ligand and regulator of germ tube formation.

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