Trichophyton mentagrophytes - Infection and Immunity - American ...

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Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by ... dihydrotestosterone were less effective inhibitors; other steroid hormones that ...
Vol. 52, No. 3

INFECTION AND IMMUNITY, June 1986, p. 763-767 0019-9567/86/060763-05$02.00/0 Copyright C 1986, American Society for Microbiology

Progesterone Binding and Inhibition of Growth in Trichophyton mentagrophytes GERTRUD SCHAR,"2 E. PRICE STOVER,3 KARL V. CLEMONS,"2 DAVID FELDMAN,3 AND DAVID A.

STEVENS' 2*

Divisions of Infectious Diseases' and Endocrinology,3 Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, and the Santa Clara Valley Medical Center and Institute for Medical Research, San Jose, California 951282 Received 4 December 1985/Accepted 5 March 1986 Specific binding of [3H]progesterone to cytosol of Trichophyton mentagrophytes was demonstrated. Scatchard analysis of [3H]progesterone binding showed a single class of binding sites with a dissociation constant of 9.5 x 108 ± 2.4 x 10-8 M (standard deviation) and a maximal binding capacity of 4,979 ± 3,489 fmol/mg of cytosol protein. Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by 50% at molar ratios of 10:1 and 20:1, respectively. Other steroid hormones that were tested had minimal activity, indicating binding specificity. Steroid hormone actions in T. mentagrophytes were examined in growth studies. Growth was assessed by determination of cellular ATP content. Progesterone inhibited growth in a dose-responsive manner, with a 50% inhibition concentration of 5.5 x 10-6 M. Partial recovery from inhibition occurrred after 24 to 48 h; inhibition could be enhanced by dividing the amount of added progesterone every 24 h. In the same rank order as was their relationship to each other and progesterone in binding studies, deoxycorticosterone and dihydrotestosterone were less effective inhibitors; other steroid hormones that were tested showed no consistent effect. We hypothesize that the binder described, acting as a hormone receptor, is the molecular site of action for the functional effect of the hormone. The functional effect may be related to the observed resistance of females to dermatophytosis. Results of recent studies of the interactions of fungi with mammalian hormones have demonstrated the presence of highly specific steroid-binding proteins in several fungal genera (6, 10-12, 14). Data derived from studies of Coccidioides immitis (5) and Paracoccidioides brasiliensis (15) indicate that effects of mammalian steroids mediated by the intracellular steroid-binding sites may be relevant in the development of the fungal disease. We studied the possible interaction of mammalian steroids with another class of pathogenic fungi, those causing the superficial mycoses. We present here data that indicate that the common dermatophyte Trichophyton mentagrophytes specifically binds progesterone and that progesterone exerts a biological effect on the fungus. (Portions of this study were presented at the Annual Meeting of the American Society for Microbiology [K. V. Clemons, E. P. Stover, G. Schar, D. Feldman, and D. A. Stevens, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, F55, p. 373]).

room temperature (Difco Laboratories, Detroit, Mich.) and transferred monthly. Preparation of cytosol. The initial inoculum was prepared from 2- to 4-week old confluent cultures on 7 ml of Sabouraud dextrose agar slants. The growth was harvested with a spatula and by rinsing, and then the harvest (5 ml, consisting of approximately 90% microconidia and 10% mycelial fragments) was added to 250 ml of Sabouraud dextrose broth. After 7 days of growth at room temperature on a gyratory shaker (150 rpm), mycelia were harvested by filtration and washed twice with cold saline and once with cold Tris-molybdenum homogenization medium (11). The mycelial mass, which was suspended in an equal volume of homogenizing medium, was mechanically disrupted in two steps. Initial breakage was done with an electric grinding probe (Tissumizer; Tekmar, Cincinatti, Ohio) at 20,000 rpm in six to eight 15-s bursts. This suspension was broken further by vigorous agitation on a vortex mixer with glass beads (0.45 to 0.50 jim in diameter) in 15-ml conical tubes (Becton Dickinson Labware, Oxnard, Calif.) in a mixture of 4 ml of suspension plus 1.5 ml of beads. This was done by alternate disruption and cooling at 0°C, for a total agitation time of 6 min for each tube. Cell debris and beads were pelleted by centrifugation (1,200 x g for 4 min at 4°C). The supernatants were centrifuged (204,000 x g for 30 min at 4°C), and cytosol was recovered as the resultant supernatant. Cytosol protein concentration was determined by the Coomassie dye binding technique (1). Steroid binding. Cytosol was incubated with various concentrations of [3H]progesterone or other tritiated steroids at 0°C for 3 h. Results of pilot experiments indicated that this time interval was sufficient to achieve maximal steady-state binding. The final sample volume for all experiments was 0.5 ml. Bound hormone was separated from free hormone by the addition of 0.5 ml of dextran-coated charcoal (0.5% Norit A,

MATERIALS AND METHODS Materials. Tritiated steroids (progesterone, 51 Ci/mM; corticosterone, 47 Ci/mM; estradiol. 47 Ci/mM; dihydrotestosterone [DHT], 60 Ci/mM), were purchased from Amersham Corp. (Arlington Heights, Ill.). Radioinert steroids were obtained from Steraloids (Wilton, N.H.). Reagents for measuring ATP were purchased from The 3M Co. (St. Paul, Minn.). Other reagents were purchased from Sigma Chemical Co. (St. Louis, Mo.), unless noted otherwise. Fungi. Clinical isolates of T. mentagrophytes (isolates 5 and 6) were maintained on Sabouraud dextrose agar slants at *

Corresponding author. 763

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FIG. 1. Equilibrium analysis of [3H]progesterone binding in T. mentagrophytes cytosol. (A) Isotherm of total, specific, and nonspecific binding at various [3H]progesterone concentrations. (B) Scatchard analysis of the specific binding data taken from the isotherm. The dissociation constant in this study was 6.8 x 10-8 M, and the binding capacity was 8,176 fmol/mg of cytosol protein (r = - 0.98). [3H]Prog bound = [H3]progesterone bound, in femtomoles per milligram of protein. Bound/free expresses [3H]progesterone bound/free. In panel B, [3H]progesterone bound is in femtomoles per milliliter.

0.05% dextran) at 0°C in homogenization medium minus glycerol. The charcoal suspensions were mixed with a vortex mixer and kept at 0°C for 12 min. The samples were then centrifuged (3,300 x g) for 7 min, and a 0.25-ml fraction of the supernatant was removed for measurement of total bound radioactivity. Nonspecific binding was determined in all experiments by replicate incubation of samples with a 500-fold molar excess of unlabeled progesterone. Specific binding was calculated by subtracting nonspecific binding from total binding. Effect of hormones in vitro on growth of T. mentagrophytes. Cultures of T. mentagrophytes were grown in a synthetic liquid medium (SAAMF) (8) at 30°C for 8 days. Mycelia were washed twice in medium, suspended in medium, and homogenized with a glass tissue grinder. Turbidity was spectrophotometrically adjusted to an optical density 0.6, and a further 1:25 dilution in medium was made. The steroids to be tested were dissolved in ethanol and diluted in medium to an ethanol concentration of 1% (vol/vol). Tubes containing 0.5 ml of mycelial suspension in medium and 0.5 ml of medium with steroid (or ethanol control; final ethanol concentration, 0.5%) were incubated at 30°C. Triplicate samples for growth curve studies were sampled at the outset and at 24-h intervals. Growth was determined by ATP photometry (13), using a modified application of the LUMAC system (The 3M Co.) (E. Lefler, J. Hamilton, and D. A. Stevens, manuscript in preparation). In brief, NRB (nucleotide-releasing reagent) and LumitPM (luciferin-luciferase) were added to the washed pellet of each tube. Integrated reading with a Biocounter M 2010 (The 3M Co.) during 10-s periods yielded ATP values expressed as relative light units. In some studies, at 24 and 48 h progesterone was added to cultures in 0.01-ml volumes, half of which was appropriately concentrated hormone in ethanol, and the other half was medium. Statistics. Data were analyzed statistically by the Student t test and Pearson correlation coefficient (r), where indicated. RESULTS

Survey of T. mentagrophytes cytosol for a steroid-binding site. To determine whether a steroid-binding site was present in T. mentagrophytes cytosol, portions of cytosol were

incubated with substantial concentrations (1.3 x 10-7 M) of various tritiated steroids, and specific binding was determined. [3H]progesterone showed a large number of specific binding sites (1,130 fmol/mg of protein), whereas [3H]corticosterone had a small umber of binding sites (90 fmol/mg of protein). Total binding of [3H]estradiol and [3H]DHT was not significantly different from nonspecific binding, indicating no detectable specific binding sites at the concentration of hormone used. Equilibrium analysis of [3H]progesterone binding. The equilibrium binding characteristics of the T. mentagrophytes isolate 5 binding site for progesterone were further examined by incubating cytosol with various [3H]progesterone concentrations at 0°C for 3 h. The binding isotherm (Fig. 1A) shows total, specific, and nonspecific binding as a function of ligand concentration. Specific binding was saturable and nonspecific binding was linear and generally less than 25% of total binding. Scatchard analysis of the specific binding data (Fig. iB) revealed a single class of binding sites with an apparent dissociation constant of 6.8 x 10-8 M and a binding capacity of 8,176 fmol/mg of protein. The mean ± standard deviations for three such experiments were 9.5 x 10-8 ± 2.4 x 10-8 M for the dissociation constant and 4,979 ± 3,489 fmol/mg of cytosol protein for the binding capacity. Identical studies were carried out on cytosol from another isolate of T. mentagrophytes, isolate 6, and the results were essentially the same. Specifity of the progesterone-binding site. The specificity of the T. mentagrophytes-binding site was examined by determining the ability of a variety of radioinert steroid hormones to compete for [3H]progesterone binding (Fig. 2). Progesterone proved to be the most potent competitor, inhibiting binding by 50% at a molar ratio of progesterone/ [3H]progesterone of 1. Deoxycorticosterone (DOC) inhibited binding by 50% at a molar ratio of DOC/[3H]progesterone of 10, and DHT inhibited binding by 50% at a molar ratio of 20. The other steroids tested were all much less active competitors. The relative potency of all steroids tested was progesterone > DOC > DHT > deoxycortisol = corticosterone > hydrocortisone > dexamethasone > estradiol. Effect of hormones on growth of T. mentagrophytes. Steroids utilized in the binding studies were assessed for their effect on T. mentagrophytes isolate 5 growth. Progesterone consistently exhibited a strong inhibitory effect on the

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FIG. 2. Specificity of the fungal binder studied by competition analysis. Cytosol was incubated at 0°C for 3 h with 1.3 x 10- M [3H]progesterone with or without the indicated concentrations of competitor. Binding in the absence of competitor was taken as 100%. Percent binding remaining is [3H]progesterone bound, as a percentage of the control. Results are means of four to six determinations. Abbreviations: PROG, progesterone; DOC, deoxycorticosterone; DHT, dihydrotestosterone; S, deoxycortisol; B, corticosterone; E2, estradiol; Dex, dexamethasone; F, cortisol.

growth of T. mentagrophytes. The results of a representative dose-response experiment are given in Fig. 3, showing a 50% inhibitory concentration of 5.5 x 10-6 M. Similar inhibition was demonstrated with T. mentagrophytes isolate 6. Growth inhibition of T. mentagrophytes isolate 5 by progesterone was also demonstrated in Sabouraud broth. In a further series of experiments, 10-6 M progesterone had no consistent effect, while 10-7 and 10-8 M progesterone slightly stimulated growth (data not shown). The other steroids that exhibited binding activity were also tested for their effects on T. mentagrophytes growth, and the results were compared with those obtained for progesterone. When the steroids (DOC, DHT, progesterone) 100 -J 0 r

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PROGESTERONE CONCENTRATION (M) FIG. 3. Dose-response of progesterone on T. mentagrophytes growth. Data are expressed as percent growth (+standard deviation) compared with concurrent ethanol control after 48 h of incubation. Progesterone concentrations, in micromoles, were 1.3, 6.5, 13, 26, 65, and 130; 50% inhibition was achieved by 5.5 x 10-6 M.

3.0 48 24 72 INCUBATION TIME (HOURS) FIG. 4. Growth of T. mentagrophytes. Inhibition by progesterone (PROG), DOC, and DHT. Data are expressed as log relative light units (RLU) (+standard deviation) of culture samples at 0, 24, 0

48, and 72 h of incubation.

were tested at the same concentration (6.5 x 10-5 M), inhibition of growth occurred in the same rank order as the binding affinity (Fig. 4). In addition to the overall growth inhibition by progesterone, an actual loss of viability (killing) was suggested in the first 24 h. At all time points inhibition by progesterone versus that by DOC and by DOC versus that by DHT was significant at P < 0.001 and P < 0.02,

respectively. Because the time-effect curve (Fig. 4) suggests a recovery from the inhibitory effect with time, we investigated whether the inhibitory effect might be potentiated by the addition of fresh hormone. In Fig. 5 it is shown that dividing the hormone and adding it at 24-h intervals increases the potency of the inhibitory effect. In this study three dosage regimens were compared: A, single dose of 3.9 x 10-5 M; B, single dose of 1.3 x 10-5 M, each added at the outset; C, 1.3 X 10-5 M added at the outset and again at 24 and 46 h. These concentrations were selected, based on the preceding studies, to be close to the threshold of the hormone effect. If the effects were too profound, differences between single and multiple regimens could be missed. The repetitive addition of hormone (regimen C) sustained the inhibition (93.7%) compared with administration of a single dose (regimen B; 61.9%; P < 0.001). Furthermore, although the time x concentration curve with regimen A was 1.5 times that with regimen C, the latter regimen was more strongly inhibitory (93.7 versus 85.5%; P < 0.005), suggesting decay of extracellular hormone in this milIeu or that metabolism of progesterone by the fung"us allows repetitive additions of fresh hormone to have the greater effect. The next experiment was an important control to demonstrate that the loss of progesterone potency did not occur in the absence of the fungus. Progesterone was incubated under experimental conditions identical to those described

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oS 3.5 48 72 0 24 INCUBATION TIME (HOURS) FIG. 5. Growth of T. mentagrophytes. Effect of repeated progesterone (PROG) addition. Data are expressed as log relative light units (RLU) (±standard error of the mean). Three regimens were studied: A, 3.9 x 10-' M added at 0 h (To); B, 1.3 x 10-i M added at 0 h; C, 1.3 x 1i-O M added at 0, 24, and 46 h. A control is also shown.

the other steroids that were studied had no consistent effect. We hypothesize that the binder demonstrated is the molecular site of action for the functional effect shown for the hormone. Growth inhibition of T. mentagrophytes by progesterone was previously reported by Campillo et al. (2) and Capek and Simek (3). In the first of these reports, the concentration used was not clearly stated, and in the latter study, a higher concentration (3.2 x 10' M) than those we studied was used. In these two studies, as well as in a third (4), it was reported that DOC inhibits the growth of T. mentagrophytes. In the first two studies it was found that DOC is more inhibitory than progesterone, which is in contrast to our findings, though where stated, the concentrations studied (3.2 x 10-4 to 7.6 x 10-4 M) were again higher than those we used. Moreover, the methods that were used previously (radial growth on agar, dry weight) were less sensitive than the method that we used. In addition, methods which use dry weight may produce erroneous results because of incorporation of steroid into the organism (7). Results of our growth studies with progesterone, in contrast to those with other methods used previously, also make it possible to suggest that more than growth inhibition is occurring, at least initially. One interpretation of the rapid loss of cellular ATP that was demonstrated is that cellular integrity is lost, possibly reflecting cell death. We suggest that the hormone binding implies the existence of a primitive hormone-receptor system in T. mentagrophytes, as has been suggested to exist in other fungi (5, 6, 10-12, 14, 15). A similar system thus would appear to have

CONTROL PROG 1.3x105M preincubated

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above, but without the fungus (Fig. 6). Progesterone in ethanol and medium, sufficient to give a final concentration of 1.3 x 10' M when mixed with the culture, was incubated at the culture temnperature of 30°C for 48 h prior to addition to the culture. The subsequent effect on the fungus was the same as that produced by preincubating the diluting medium alone at 30°C for 48 h and adding the progesterone in ethanol to give the same final concentration at the time of addition to the fungal culture. This suggests that the loss of inhibitory activity is due to metabolism of hormone by the fungus and not to decay of the added hormone or binding to medium constituents or the vessel during incubation with the fungus. Hydrocortisone tested at concentrations of 10-4 to 10-9 M showed non-dose-responsive stimulation of growth (29 to 50% for 10-5 to 10-8 M versus control; for each, P < 0.05) during the first 24 h of incubation; however, this effect was lost with prolonged incubation. Corticosterone and triamcinolone acetonide at concentrations of 10-4 and 10-6 M and dexamethasone at 10-6 M had no significant effect on the growth of T. mentagrophytes (data not shown).

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DISCUSSION We have shown here that progesterone specifically binds to T. mentagrophytes cytosol and have characterized that binding. A single class of binding sites appears to be involved. DOC and DHT compete somewhat for this site, but other steroids are minimally active. We also show that progesterone inhibits T. mentagrophytes growth, as do DOC and DHT, in proportion to their binding activity, whereas

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INCUBATION TIME (HOURS) FIG. 6. Growth ofT. mentagrophytes. Effect of preincubation of progesterone (PROG). Data are expressed as described in the legend to Fig. 5. Culture medium was incubated at 30°C for 48 h without fungus and with (1.3 x 10-5 M) or without progesterone. Progesterone (1.3 x 10-5 M) was added to the latter at the end of 48 h, and then fungal inoculum in medium was added to both of these and to the control medium incubated under the same conditions without progesterone.

VOL. 52, 1986

been conserved in the evolution of eucaryotic organisms from single-celled to mammalian species. The fungal binding moiety presumably binds an endogenous fungal hormone, one that is possibly structurally related to mammalian progesterone. The importance of this system in the cell biology of the fungus is unknown, but results of our studies suggest a possible growth-regulatory role. The binding of mammalian progesterone may have consequences for the interaction between T. mentagrophytes and the mammalian host in dermatophyte infection. It is known that the peak incidence of these infections is 5 times more common in males than in females and the incidence in females rises after menopause (9), which suggests that progesterone may inhibit the growth of the fungus in vivo. The 50% effect on function (growth) we have demonstrated here occurs at a much higher concentration compared with circulating progesterone levels in women or with the concentration at which 50% of the binding sites are filled by hormone, although a precise concordance cannot be expected because of the unknown effect of variables in the in vitro system (nutrients, oxygenation, etc.). Taken together, the data in Fig. 5 and 6 suggest that another factor influences this system. In our in vitro experitnents, hormone was added at the beginning of the culture period; when hormone was replenished, the overall effect became more powerful. The reintroduction of fresh hormone into the microenvironment more closely resembles the in vivo situation in the skin. The loss of hormone activity during incubation required the presence of the fungus. Results of these experiments suggest, and preliminary studies (Clemons et al., Abstr. Annu. Meet. Am. Soc. Microbiol., 1985), including high-performance liquid chromatography, further suggest that the fungus is metabolizing the hormone, thereby possibly lessening its effect. These findings support the preliminary report by Capek and Simek (3) that T. mentagrophytes metabolizes progesterone to 15-a- and 15-p-hydroxyprogesterone. The metabolic activity of the organisms explains, at least in part, the relatively high progesterone concentration required for a functional response. This factor could operate in vivo, which is a consideration of the question of whether circulating levels of progesterone inhibit 7T. mentagrophytes growth. That high concentrations of progesterone are required to elicit the action does not detract frotn the importance for fungal biology of the finding of inhibition. The requirement for high concentrations may result from extraneous reasons such as metabolism of the steroid and poor entry of hormone into cells. The importance of the finding is that whether or not inhibition occurs in humans, the effect appears to relate to hormone binding by the three steroids that induce the function and is not the result of a nonspecific toxic action. Thus, the biologic importance is related to the finding of a functional response to added hormones that bind to putative receptors within the fungus.

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ACKNOWLEDGMENT This study was supported by Public Health Service grant AI-20409 from the National Institutes of Health.

LITERATURE CITED 1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utiiizing the principle of protein dye binding. Ann. Biochem. 72:248-254. 2. Campillo, C. C., D. Balandrano, and A. Galarza. 1961. Antimicrobial properties of 21, 21 dimethoxy progesterone and other progesterone analogues. J. Bacteriol. 81:366-375. 3. Capek, A., and A. Simek. 1971. Effect of steroids on dermatophytes. Folia Microbiol. 16:299-302. 4. Chattaway, F. W., J. D. Townsley, and A. J. E. Barlow. 1959. Effect of steroids and related compounds on the growth of dermatophytes. Nature (London) 184:1731-1732. 5. Drutz, D., M. Huppert, S. H. Sun, and W. L. McGuire. 1981. Human sex hormones stimulate the growth and the maturation of Coccidioides immitis. Infect. Immun. 32:897-907. 6. Feldman, D., Y. Doj A. Burshell, P. Stathis, and D. S. Loose. 1982. ASn estrogen-binding protein and endogenous ligand in Saccharomyces cerevisiae: possible hormone receptor system. Science 218:297-298. 7. Ghannoum, M. A., S. Mudher, and G. Burns. 1985. Incorporation of dexamethasone by Candida albicans. Microbios

42:103-109. 8. Hoejrich, P. D., and P. D. Finn. 1972. Obfuscation of the activity of antifungal antibiotics by culture media. J. Infect. Dis. 126:353-361. 9. Jones, H. E. 1983. Superficial fungus infections of the skin, p. 966-978. In P. D. Hoeprich (ed.), Infectious diseases, 3rd ed. Harper & Row, Publishers, Inc., Philadelphia, Pa. 10. Loose, D. S., D. J. Schurman, and D. Feldman. 1981. A corticosteroid binding protein and endogenous ligand in Candida albicans indicating a possible steroid-receptor system. Nature (London) 293:477-479. 11. Loose, D. S., D. A. Stevens, D. J. Schurman, and D. Feldman. 1983. Distribution of a corticosteroid-binding protein in Candida and other fungal genera. J. Gen. Microbiol. 129:2379-2385. 12. Loose, D. S., E. P. Stover, A. Restrepo, D. A. Stevens, and D. Feldman. 1983. Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis. Proc. Natl. Acad. Sci. USA 80: 7659-7663. 13. Odds, F. C. 1980. Laboratory evaluation of antifungal agents: a comparative study of five imidazole derivatives of clinical importance. J. Antimicrob. Chemother. 6:749-761. 14. Powell, B. L., D. Drutz, M. Huppert, and S. H. Sun. 1983. Relationship of progesterone- and estradiol- binding proteins in Coccidioides immitis to coccidioidal dissemination in pregnancy. Infect. Immun. 40:478-485. 15. Restrepo, A., M. E. Salazar, L. E. Cano, E. P. Stover, D. Feldman, D. A. Stevens. 1984. Estrogens inhibit mycelium-toyeast transformation in the fungus Paracoccidioides brasiliensis: implications for resistance of females to paracoccidioidomycosis. Infect. Immun. 46:346-353.