Proc. NatL. Acad. Sci. USAVol. 80, pp. 7659-7663, December 1983Medical Sciences

Estradiol binds to a receptor-like cytosol binding protein andinitiates a biological response in Paracoccidioides brasiliensis

(steroid hormone action/fingus/paracoccidioidomycosis)

DAVID S. LooSE*, E. PRICE STOVER*, ANGELA RESTREPOt, DAVID A. STEVENS**, AND DAVID FELDMAN*§*Depajtment of Medicine, Stanford University School of Medicine, Stanford, CA 94305; tCorporacion De Investigaciones Biologicas, Medellin, Colombia; andtSanta Clara Valley Medical Center and Institute for Medical Research, San Jose, CA 95128

Communicated by Dale Kaiser, September 8, 1983

ABSTRACT Paracoccidioidomycosis, a disease caused byParacoccdioides brasilensis, which is endemic to Latin America,is much more common in men than women, suggesting a role forhormonal factors. We recently showed that two other yeasts pos-sess steroid binding proteins and postulated that these receptor-like molecules represented a mechanism by which the hormonalmilieu of the host might influence an infecting pathogen. There-fore, we examined P. bralensis for a sex steroid binding protein.Because tritiated steroids rapidly dissociated from the other fun-gal binding proteins, we developed a fast binding method withSephadex G-50 microcolumns speeded by centrifugation. Thismethod detected specific binding of [3H]estradiol in P. braWiensiscytosol. Other tritiated steroid hormones, including testosteroneand corticosterone, failed to exhibit specific binding. Scatchardanalysis of [3H]estradiol binding showed an apparent dissociationconstant (Kd) of 1.7 x 10-8M and a maximal binding capacity (No)of 235 fmol/mg of protein. Susceptibility to trypsin indicated thebinding site was protein in nature. The protein had a Stokes radiusof -32 A by HPLC exclusion column and a sedimentation coef-ficient of 4.4 S by sucrose gradient, consistent with an apparentMr of =60,000. Competition experiments revealed that estrone,estriol, and progesterone had 25% of the affinity of estradiol,whereas diethylstilbestrol, androgens, and corticosteroids had lowaffinity. Investigation of steroid hormone actions in P. brasiienisindicated that estradiol inhibited the fungal transformation frommycelial form to yeast form, the initial step of infection. This sup-pressive effect was dose-dependent and not found with testoster-one. We hypothesize that endogenous estrogens in the host, actingthrough the cytosol binding protein in the fungus, inhibit mycelial-to-yeast transformation, thus explaining the resistance of women*to paracoccidioidomycosis.

Paracoccidioides brasiliensis is a pathogenic fungus, the etio-logic agent of paracoccidioidomycosis (South American blas-tomycosis). Although paracoccidioidomycosis is 13-87 times morecommon in men than women (1), contact with P. brasiliensis isessentially the same for the two sexes (2). The possibility hasbeen considered that the hormonal milieu of the host mightdirectly influence P. brasiliensis, affecting its pathogenicity (1,3). Although this hypothesis is speculative, our recent findings(4-6) suggest a basis for a mechanism by which host hormonescould affect an invading pathogen and alter its infectious prop-erties. We have demonstrated the existence of intracellularproteins in two fungal genera that bind vertebrate steroid hor-mones with high affinity and specificity. Candida albicans pos-sesses a protein that binds corticosterone and progesterone (4,5), and Saccharomyces cerevisiae contains a different proteinthat selectively binds estrogens (6). We have postulated thatthese macromolecules may represent primitive hormone re-

ceptors in fungi. These proteins appear not only to bind en-dogenous fungal ligands but also to bind vertebrate steroid hor-mones, a fact we have exploited in selecting the radioprobes forthese studies. Therefore, we considered the possibility that P.brasiliensis could possess either an androgen or an estrogenbinding protein which, when occupied by the appropriate hostsex steroid, might result in altered pathogenicity, thus explain-ing the predominance of infections in males. The data to bepresented in this report demonstrate that P. brasiliensis doescontain an estrogen binding protein. We also show that estra-diol inhibits the fungal, transformation from mycelial form toyeast form (mycelial-to-yeast transformation), the initial step inthe establishment of infection (7). Although the concordance ofbinding in vitro and bioactivity of hormones in culture is notexact, the findings support our hypothesis-that pathogenic fungihave the capacity to respond to-the hormonal environment ofthe host through receptor-like steroid binding proteins.

MATERIALS AND METHODSMaterials. Tritiated steroids were purchased from Amer-

sham. Nonradioactive steroids were purchased from Steraloids(Wilton, NH). All chemicals were reagent grade purchased fromSigma unless otherwise indicated.

Yeast Cultures. The binding studies used a clinical isolate ofP. brasiliensis from a male Colombian patient with paracoccid-ioidomycosis. From a stock culture maintained on agar, yeast-phase cells were inoculated into 5 ml of modified McVeigh-Morton liquid' medium (8). After 3-4 days growth at 350C ona gyratory shaker (200 rpm), the contents were added to 300 mlof fresh medium and incubated for an additional 2 wk withshaking. After 200-ml portions were harvested for bindingstudies, the remainder was added to 300 ml of fresh medium;the process was repeated weekly. Contamination of the cul-tures by other organisms was excluded by subculturing ontoblood agar plates before harvesting. Cells were harvested bycentrifugation and washed three times with saline. Yeasts weredisrupted at 40C by vigorous agitation with 250- to 300-gm glassbeads on a Vortex mixer for about 15 min in a homogenizationmedium containing 250 mM sucrose, 10 mM Tris, 1.5 mMEDTA, 12 mM monothioglycerol, 10 mM Na2MoO4, and 10%glycerol (pH 7.8). Tubes were -kept cold by immersion in an iceslurry. Cytosol was prepared by 204,000 X g ultracentrifuga-tion for 30 min.

Binding Studies. Steroid binding assays were performed bya modification of previously reported techniques (4-6). Cytosol(1-3 mg/ml) was incubated with tritiated steroids for 3 hr atOOC, a period sufficient to obtain apparent equilibrium. Non-specific binding was determined in all experiments by replicate

Abbreviations: DES, diethylstilbestrol.§ To whom reprint requests should be addressed.

7659

The publication costs of this article were defrayed inpart by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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incubation of samples with a 500-fold molar excess of the ap-

propriate radioinert steroid. Bound hormone was separated fromfree hormone by using a gel exclusion microcolumn of G-50fine Sephadex (Pharmacia). Columns were prepared by fillinga 3-ml plastic syringe barrel (Monoject, Sherwood Medical In-dustries, Deland, FL) with Sephadex preswelled in 100 mMTris/3 mM CaC12, pH 6.9. Columns were allowed to drain, andadditional slurry was added to entirely fill the syringe. The mi-crocolumns were placed in 13-mm tubes for centrifugation at100 X g (average) for 2-3 min at O'C. A centrifugation step priorto addition of the sample removed most of the buffer from thecolumn, preventing protein dilution during the actual separa-

tion. Cytosol samples of 200 pil were then loaded onto the col-umns, which were centrifuged; the eluate, containing protein-bound hormone, was collected.

Cytosol protein concentration was measured by the BradfordCoomassie-dye binding technique (9) with 80:20 human gamma

globulin/bovine serum albumin serving as a standard.Enzymatic and Thermal Studies. Cytosol was prepared as

described above in standard homogenization medium. DNaseand RNase (Worthington) were tested in the presence of 6 mMMgCl2. Phospholipase A2 (Sigma) was tested in the presence of2 mM CaC12. Neuraminidase (Millipore) and trypsin (1:200;Difco) were tested in standard homogenization medium.

Hydrodynamic Properties. Cytosol preincubated with 130nM [3H]estradiol for 3 hr at 0°C was analyzed by HPLC witha Varian model 5000 equipped with an ice water-jacketed TSK-3000 gel exclusion column as described (10). Labeled cytosolwas injected onto the column and eluted in 0.2 M phosphatebuffer (pH 7.4).

Effect of Hormones in Vitro on P. brasilensis. Isolates of P.brasiliensis (mycelial form) were incubated in McVeigh-Mor-ton medium at 20-25C. Homogenization was performed in a

laboratory blender in one or two 5-sec bursts until the inoculumconsisted, by microscopic observation, of well-dispersed hy-phal fragments. Turbidity was adjusted to equal that of tube 5on McFarland scale (11). A microculture system previously de-scribed (12) was used for the assays. In brief, the steroids to betested were diluted in ethanol and mixed at 50°C with Mc-Veigh-Morton medium and agar or agarose. Controls receivedidentical concentrations of ethanol (1 ,uM hormone contained3.0% ethanol; 0.1 puM hormone contained 0.3% ethanol, etc.).Ethanol at these concentrations was shown not to affect trans-formation compared to 0% ethanol controls. The agar mixtureswere allowed to gel at 4°C, and 1-cm2 blocks were transferredaseptically to a microscope slide. Inoculum (0.01 ml) was addedto the blocks, a coverslip was applied, and the slide was in-cubated at 36°C for 5 days. Two microcultures were set up ateach concentration in each experiment for each isolate studied.The blocks were examined microscopically, 100 cells per mi-croculture, and the percentage of mycelial fragments untrans-formed or transformed to yeast-form cells (7) were enumerated.The range of transformation in all experiments for control cul-tures was 35-78%.

RESULTSThe Sephadex Microcolumn Chromatography Method. Our

experience with other fungal steroid binding proteins (4-6) hasbeen that the steroid-protein complexes have a relatively fastdissociation rate. In order to circumvent problems of receptor-ligand dissociation during measurement, we- developed a rapid(2-3 min) and simple separation system by using Sephadex G-50 microcolumns (3-ml) with sample elution speeded by cen-

trifugal force. Similar techniques have been described (13, 14).These columns provided an excellent separation of macromol-

5,000

10

4.')

0.4

Eluate, ml

0

c)

e-CD

FIG. 1. Elution characteristics of the centrifugal microcolumns.Syringe barrels were filled with a slurry of Sephadex G-50 and pre-centrifuged at 100 x g for 5 min. Samples of various volumes contain-ing either 1 x 106 dpm of [3H]estradiol (0) or 1.0 mg of bovine serumalbumin (0) were layered on the columns and centrifuged again. Eachpoint is the mean of triplicate determinations.

ecules from the free steroids (Fig. 1). No free steroid was de-tected in the eluate until a sample volume of =400 p1 was used.An increase in the mass of the free steroid added to the columnby a factor of 500 did not affect this performance, indicatingthat the columns were not being overloaded by the smalleramounts of steroid used in the studies to be described here.Protein recovery from 200-pl sample volumes of albumin so-lutions or cytosol was :70% with a generous amount of "head-room" after the protein and before the appearance of free ste-roid. With P. brasiliensis cytosol, the method yielded bindingresults superior to those obtained with either Sephadex G-50minicolumn or dextran-coated charcoal separation techniques(4-6).

Specificity of Tritiated Steroid Binding in P. brasiliensis Cy-tosol. With the microcolumn method, a series of tritiated ste-roids was tested for binding to P. brasiliensis cytosol at the sub-stantial concentration of 130 nM (Table 1). [ H]Estradiol wasby far the most active ligand, with specific binding of =200 fmol/mg of cytosol protein. Some specific binding was detected with[3H]progesterone and [3H]diethylstilbestrol ([3H]DES)-60 and42 fmol/mg of protein, respectively. Only a trace of bindingwas detected with [3H]dihydrotestosterone and [3H]testoster-one, and no [3H]corticosterone binding was seen (that is, therewas no significant difference between total and nonspecificbinding). [3H]Progesterone binding was completely eliminatedby preincubation of cytosol with radioinert estradiol. These data

Table 1. Binding of various hormonal radioprobes to cytosolprepared from P. brasiliensis

Specific binding,Radioprobe fmol/mg

Estradiol 203 ± 44Progesterone 60 ± 7DES 42± 9Dihydrotestosterone 23 ± 7Testosterone 4 ± 3Corticosterone 0

Cytosol was incubated with 130 nM of each tritiated radioprobe withor without a 500-fold molar excess of the appropriate radioinert hor-mone for 3 hr at 0MC. Bound hormone was separated from free hormonewith microcolumns. Results are means ± SEM of at least four deter-minations.

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suggest the presence of a single estrogen binding site with somecrossreactivity of the other sex steroids. However, the pres-ence of a separate progesterone or DES binding site has notbeen completely excluded.

Time-Course of Tritiated Estrogen Binding. We proceededto further characterize the tritiated estrogen binding moiety inP. brasiliensis. Cytosol was prepared and incubated with 130nM [3H]estradiol for various time intervals, and specific bind-ing was determined (Fig. 2A). Binding was fairly rapid at 00C,reaching maximal levels at about 90 min and remaining stablefor several hours. After overnight (22 hr) incubations, some de-cline in [3H]estradiol binding was usually noted. Therefore, mostof the studies presented in this report were conducted at 00Cfor 3 hr.

To determine the dissociation rate (kd) of the bound [3H]es-tradiol, a 500-fold excess of radioinert estradiol was added tothe prebound cytosol to eliminate the forward reaction ([3H]es-tradiol binding). Samples of cytosol were removed and assessedfor specific binding sequentially over time. The logarithmic plotof the binding as a function of time is shown in Fig. 2B. Dis-sociation appears to be first-order with aid of 2.4 x 10-2 mind1,the time for half-dissociation then being 29 min. Binding in con-trol samples was stable over this time interval.

Equilibrium Analysis of Tritiated Estrogen Binding. Theequilibrium binding characteristics of the P. brasilwnsis bind-ing site were examined by studies at multiple tritiated estrogenconcentrations at 0C for 3 hr. The isotherm (Fig. 3A) showstotal, nonspecific, and specific binding as a function of ligandconcentration. Note the saturability of the specific binding andthe relatively low nonspecific binding (<20%). Scatchard anal-ysis of the specific binding data (Fig. 3B) shows that the datafit well to a straight line, suggesting a single class of noninter-acting binding sites. The mean values for six such experimentswere 1.7 ± 0.3 X 10-8 M for the apparent dissociation constant(Kd) and 235 ± 73 finol/mg of protein for the binding capacity.

Specificity of the Estrogen Binding Site. Studies were per-formed next to assess the specificity of the estrogen binding siteby determination of the ability of a variety of radioinert ligandsto compete for [3H]estradiol binding (Fig. 4). Estradiol provedto be the most potent competitor, achieving 50% inhibition of

0 1 2 3 18Incubation time, hr

700600500

400

300

200,1

u *0 4 8 12 16Dissociation time, min

FIG. 2. Time course of association and dissociation of the [PH}es-tradiol-fungal binder complex. (A) Association. Cytosol was incubatedat 00C with 130 nM [3Hestradiol with or without a 500-fold molar ex-cess of radioinert estradiol to assess nonspecific binding. Specific bind-ing data are plotted at the indicated incubation intervals. (B) Disso-ciation. After labeling cytosol with 130 nM [3Hlestradiol for 90 min at00C, a 500-fold molar excess of radioinert estradiol was added to theincubation mixture (0 time). Aliquots of cytosol were removed at theindicated intervals, and specific bindingwas determined. Thehalf-timefor dissociation was 29 min in this experiment.

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130 0 100 200

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FIG. 3. Equilibrium analysis of [3Hlestradiol binding in P. brasi-liensis. (A) Isotherm oftotal (o), specific (A), and nonspecific (A) bindingat various [MHlestradiol concentrations. (B) Scatchard analysis of thespecific binding data taken from the isotherm. TheKd in this study was1.5 x 108M and the binding capacity was 210 finol/mg of cytosol pro-tein.

binding-with a molar ratio of estradiol/[3H]estradiol of 2.5. Es-trone and estriol displayed about 25% of the apparent affinityof estradiol. Note that the synthetic estrogen DES was a weakcompetitor. In studies with mammalian estrogen receptors, thissynthetic compound usually has been found to exhibit approx-imately the same affinity as that of estradiol. Of the nonestro-gen steroids tested, progesterone was the most potent of thecompetitors, having approximately the same affinity as that ofestrone and estriol for the fungal binder. The androgens dihy-drotestosterone and testosterone were weak competitors.

Stability of the Fungal Binding Site to Enzymatic and Ther-mal Destruction. To test thermal stability of the binding site,cytosol was prepared and incubated at 0, 37, and 560C for 30min and then chilled to 0C prior to assay of [3H]estradiol bind-ing. Not only did the fungal binding site appear to be stable totreatment at 370C but also an increase in the amount of specific

125-v ..

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0 5\ DESH,0 f~~~~~~rog.2 50 \

If 25

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FIG. 4. Specificity of the fungal binder by competition analysis.Cytosol was incubated with 130 nM [3H]estradiol with or without theindicated concentration of competitor. Binding in the absence of com-petitor (245 fmol/mg) was taken as 100%. Results are means ± SEMof four to seven determinations. E2, estradiol; E1, estrone; E3, estriol;Prog, progesterone; DHT, dihydrotestosterone; and T, testosterone.

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g100

00

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00 37 56 DNase RNase PLase Mal- Tryp

OC NEt

FIG. 5. Stability of the P. brasiliensis binding site to thermal andenzymatic treatment. Cytosol was treated at 37 or 56TC alone or withvarious enzymes (100 Mig/ml) orN-ethylmaleimide (20 mM) at 37TC for30 min. After this treatment period, [3H]estradiol binding was assayedat 000 for 3 hr. The results are presented as a percentage ofthe bindingfound in the control samples at 3700. PLase, phospholipase A2; MaINEt,N-ethylmaleimide; Tryp, trypsin.

binding was consistently observed (Fig. 5). In additional stud-ies not detailed here, the increase in binding appeared to bedue to the appearance of a second, low-affinity binding site.The [3H]estradiol binding proved to be remarkably stable, even

to heating at 56°C. However, we have not yet resolved the pro-portion of high- or low-affinity sites left after 56°C heating.The effect of treatment of fungal cytosol with a variety of

enzymes was also investigated. All treatments were carried outfor 30 min at 37°C. DNase, RNase, and phospholipase A2 hadlittle effect on the amount of [3H]estradiol bound, whereastrypsin abolished the binding (Fig. 5). N-Ethylmaleimide (20mM) significantly reduced [3H]estradiol binding. We infer fromthese studies that the fungal binder is, at least in part, a proteinthat contains sulfhydryl groups necessary for binding.

Hydrodynamic Properties. Fungal cytosol was incubated with130 nM [3H]estradiol for 3 hr at 0°C. After free steroid was re-moved by microcolumn, the sample was injected onto an HPLCgel exclusion column. Fig. 6 shows the elution profile of [3H]-

a~or..0 1,00c

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Table 2. Effect of sex hormones on mycelial-to-yeasttransformation of P. brasiliensis

Mycelial-to-yeasttransformation,

Hormone nM % of controlEstradiol 0.1 71 ± 10*

10 33 ± 10*1,000 19 ± 10*

Testosterone 0.1 106 ± 2910 112±29

1,000 84 ± 29DES 0.1 85 ± 30

10 54±30*1,000 37 ± 30*

Three patient isolates were studied for mycelial-to-yeast transfor-mation. Data are expressed as the mean percentage transformationcompared to the appropriate control isolate (ethanol vehicle), whichtransformed between 35-55%. The true mean for each hormone andconcentration lies in the given interval with 95% confidence. The val-ues not indicated by an asterisk contain the value 100 within the in.terval and, thus, do not differ from control values. The intervals arebased on standard normal theory, with a pooled estimate of standarderror for each drug.* Values different from control values with >95% confidence.

estradiol. The binding activity was eluted as a single peak be-tween the two marker proteins bovine serum albumin (Stokesradius R8, 35.9 A; Mr, 67,000) and ovalbumin (R,, 28.6 A; Mr,45,000). In studies not shown, the P. brasiliensis binding pro-tein demonstrated a sedimentation coefficient of =4.4 S by su-crose density gradient analysis performed as described (5). Al-though precise estimates of molecular weight are difficult usingthese techniques, the results are consistent with a Stokes radiusof -32 A and a Mr of =60,000.

Effect of Hormones on P. bra&liensis. A series of studies ofthe effects of sex hormones on the fungus in vitro were per-formed concurrently and will be reported in detail elsewhere.A striking finding was the inhibition of conversion of the my-celial form to the yeast form by 17,B3estradiol (Table 2). In ex-periments with three recent patient isolates, 17,3estradiol in-hibited conversion compared to control cells. Inhibition wassignificant at concentrations as low as 0.1 nM, with >50% ofthe effect detected at 10 nM. DES appeared to be less activethan estradiol, and testosterone was inactive. In preliminaryexperiments with the isolate in which the binding studies wereperformed, roughly similar results were obtained. However,the hormones appeared to be less potent with this isolate thanthe isolates shown in Table 2, requiring concentrations be-tween 10 and 100 nM estradiol to achieve significant inhibitionof mycelial-to-yeast conversion. Again, estradiol appeared to bemore active than DES, and testosterone was inactive. Furtherstudy will be required to confirm whether differences in isolatesensitivity exist.

DISCUSSIONIn these studies we have demonstrated the existence of a mac-romolecule in P. brasiliensis that binds [3H]estradiol with highaffinity and specificity. This steroid binding protein appears tobe analogous to the proteins we have described recently in twoother fungal genera. C. albicans was found to possess a proteinthat bound corticosterone and progesterone (4, 5), whereas aprotein in S. cerevisiae bound estradiol (6). In those cases, evi-

Retention time, min

FIG. 6. Hydrodynamic properties of the [3H]estradiol binding pro-tein inP. brasiliensis. Cytosol was incubated with 130nM [3H]estradiolin the absence (e) or presence (o) of a 250-fold excess of radioinert es-tradiol for 3 hr at 00C. Free hormone was removed by passage over amicrocolumn. Cytosol protein (200 pg) was injected onto a Varian HPLCequipped with a TSK-3000 gel exclusion column, previously calibratedwith a variety of proteins. Samples were collected at 0.5-min intervalsat a flow rate of 1 ml/min and assayed for radioactivity. Markers shownare bovine serum albumin (I) and ovalbumin (II).

dence for the existence of an endogenous ligand also was pro-vided, which has led us to hypothesize that some fungi mightpossess a hormone-receptor system. At present we have notyet examined whether P. brasiliensis possesses an endogenous

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ligand. In each case, however, the fungal binding proteins ex-hibit high affinity for vertebrate steroid hormones. This has ledus to speculate that certain pathogenic fungi have the capacityto respond to the hormonal environment of the host. Circu-lating host hormones, by interacting with fungal binding pro-teins, may alter fungal properties and, thus, affect pathogenici-ty. In the case of P. brasiliensis, our findings additionally provideevidence that estradiol mediates an important biological effectin the fungus. Mycelial-to-yeast transformation was inhibitedby estradiol in a dose-dependent manner. Testosterone was es-sentially inactive.

Because P. brasiliensis is known to display such a markedpreference for males, it was tempting to speculate that host es-trogens interfered with the growth of the fungus or affectedpathogenesis. The data in Table 2 support this hypothesis. My-celial-to-yeast transformation is an early and, thus, critical stepin the development of infection in an individual exposed to P.brasiliensis mycelia (7). The current findings of both an estro-gen binding protein and estradiol action to inhibit mycelial-to-yeast conversion suggest that this phenomenon is the possiblemolecular basis for the reduced infection rate in women. How-ever, other estradiol effects in vivo may additionally contributeto the resistance of females to this infection. Also, the fact thatDES does not bind to the estrogen binding protein with highaffinity and yet is a good inhibitor of transformation suggeststhat additional estrogen-mediated actions in the fungus also maybe involved. Although it has been reported (3) that estradiolinhibited P. brasiliensis growth in vitro, this occurred at ex-tremely high concentrations, well beyond the physiologic range.

Implicit in our hypothesis that estradiol mediates functionalresponses in P. brasiliensis through this estrogen binding pro-tein is the notion that the estrogen binding protein is a recep-tor-like molecule, presumably for an endogenous ligand in thefungus. The ability of mammalian sex hormones to bind to thisprotein suggests evolutionary conservation of the ligand bind-ing site.The finding of an estrogen binding protein in P. brasiliensis

provides a possible cellular mechanism by which host sex hor-mones might interact with the pathogen. A difficulty with thishypothesis is that the peak circulating levels of estradiol inmenstruating women is about 1 order of magnitude lower thanthe apparent Kd of the fungal binding site. If one considers onlythe free (i.e., not protein bound) estradiol, the discrepancy be-comes even larger. However, several factors might compensatefor this difference between apparent in vitro affinity and in vivosteroid concentration. (i) The affinity of the protein for estradiolmight actually be higher. Our in vitro measurements may notbe optimal because of the presence of endogenous ligand orother factors. Also the affinity in intact fungi or in mycelial-form

fungi may be higher; but, because of biohazard problems, wehave not yet examined these possibilities. (ii) Other estrogenssuch as estrone and estriol (as well as progesterone) also occupythe fungal binder, and effects might well be additive. (iii) P.brasiliensis may concentrate estrogens, achieving sufficientlyhigh levels to occupy the fungal binder. (iv) An estradiol-me-diated function may occur even with a small proportion of bind-ing sites occupied. At 6.5 nM [3H]estradiol, we routinely de-tected 30-80 fmol of [3H]estradiol bound per mg of P. brasiliesicytosol protein. In the experiments showing inhibition of my-celial-to-yeast transformation (Table 2), significant estradiolbioactivity could be detected at 0.1 nM.

In conclusion, this report describes a receptor-like bindingprotein in P. brasiliensis that selectively binds estrogens. In ad-dition, we have demonstrated that estradiol inhibits mycelial-to-yeast-form transformation in this pathogen. There is a strik-ing sex preference in this disease, and we believe the currentfindings support our hypothesis that circulating estrogens inthe host inhibit pathogenicity of the fungus by interacting withthis binding protein.The authors thank M. E. Salazar for assistance with the function ex-

periments and Brad Efron for assistance with the statistical analysis ofthe data in Table 2. This work was supported by National Institutes ofHealth Grants GM 28825 and AI 20409, National Science FoundationGrant INT 83-02725, and Colciencias, Colombia Grant 9714 5-3-W80.

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2. Restrepo, A., Robledo, M., Ospina, S., Restrepo, M. & Correa,A. (1968) Amer. J. Trop. Med. 17, 25-37.

3. Muchmore, H. G., McKown, B. A. & Mohr, J. A. (1972) PanAm.Health Org. Sci. Pub. 254, 300-372.

4. Loose, D. S., Schurman, D. & Feldman, D. (1981) Nature (Lon-don) 293, 477-479.

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Science 218, 297-298.7. Rippon, J. W (1980) CRC Crit. Rev. Microbiol. 8, 49-97.8. Restrepo, A. & Jimenez, B. E. (1981)J. Clin. Microbiol. 12, 279-

281.9. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.

10. Pavlik, E. J., Van Nagell, J. R., Muncey, M., Donaldson, E. S.,Hanson, M., Kenady, D., Rees, E. D. & Talwalkar, V. R. (1982)Biochemistry 21, 139-145.

11. Finegold, S. M., Martin, W J. & Scott, E. G., eds. (1978) in Bai-ley and Scott's Diagnostic Microbiology (Mosby, St. Louis, MO),pp. 488-489.

12. Restrepo, A., Cano, L. E., deBedout, C., Brummer, E. & Ste-vens, D. A. (1982)J. Clin. Microbiol. 16, 209-211.

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14. Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899.

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