May 28, 1985 - a high-affinity, low-capacity, tritiated 17I-estradiol ([3H]estradiol)-binding protein. ... purchased from the Amersham Corp., Arlington Heights, Ill.
Vol. 51, No. 1
INFECTION AND IMMUNITY, Jan. 1986, P. 199-203
0019-9567/86/010199-05$02.00/0 Copyright © 1986, American Society for Microbiology
Estradiol-Binding Proteins from Mycelial and Yeast-Form Cultures of Paracoccidioides brasiliensis E. PRICE STOVER,' GERTRUD SCHAR,2 3 KARL V. CLEMONS,2 3 DAVID A. STEVENS,2'3 AND DAVID FELDMAN'* Divisions of Endocrinology' and Infectious Diseases,2 Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, and Santa Clara Valley Medical Center and Institute for Medical Research, San Jose, California 951283 Received 28 May 1985/Accepted 30 September 1985
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, causes disease much more frequently in men than it does in women, suggesting that the hormonal milieu of the host might influence P. brasiliensis pathogenicity. We recently demonstrated that cytosol from yeast cultures of P. brasiliensis contains a high-affinity, low-capacity, tritiated 17I-estradiol ([3H]estradiol)-binding protein. Estradiol and, to a lesser degree, diethylstilbestrol (DES), inhibited the transformation of P. brasiliensis cultures from the mycelial to the yeast form, an event critical to the establishment of infection. Our current studies demonstrated a somewhat higher affinity (apparent dissociation constant [Kd], -6 to 12 nM) of the estrogen-binding protein for [3H]estradiol than was previously described for yeast cytosol. The presence of both high- and low-affinity estrogen-binding sites in yeast-form P. brasiliensis cytosol was detected after warming the cytosol to 37°C. The high-affinity protein was labile to further heating (56°C), although the low-affinity protein was stable. Additional experiments demonstrated the presence of an estrogen-binding protein in cytosol prepared from mycelial-form P. brasiliensis. This estrogen-binding protein had a slightly lower affinity for [3H]estradiol (Kd 13 nM), and its cytosol contained somewhat fewer binding sites (-78 fmol/mg of protein) than did yeast-form P. brasiliensis cytosol. Of particular interest was the finding that DES, a weak competitor for [3H]estradiol binding in yeast cytosol, displaced [3H]estradiol from the mycelial-form binding moiety. DES had a 50- to 100-fold-lower affinity for the [3H]estradiol-binding protein than did estradiol, consistent with its lower bioactivity in the mycelial-to-yeast-form transformation studies. The current results lend further support to our hypothesis that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast-form transformation, thus explaining the resistance of women to paracoccidioidomy-
cosis.
Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is indigenous to regions of South America. Although this organism causes disease 13 to 87 times more frequently in men than it does in women (3), the results of skin test studies indicate that contact with the pathogen is equal for the two sexes (8). Moreover, there is no sex-based difference in the disease rate of prepubertal individuals (7). This striking epidemiology led us to hypothesize that the hormonal environment of the host might directly affect P. brasiliensis and alter its pathogenicity. We reported the presence of a high-affinity, receptorlike binding protein for estradiol in cytosol from yeast cultures of P. brasiliensis (5). In addition, this binding protein was shown to be present in three additional clinical isolates (9). We presented evidence that estradiol can also regulate an important biological event in P. brasiliensis. Exposure of the fungus to physiologic concentrations of estradiol inhibited the transformation from the mycelial to the yeast form (5, 9), a critical event in the establishment of infection (10). We believe these experiments documented a possible molecular basis for the marked resistance of women to paracoccidioidomycosis. In this paper, we describe the results of further experiments with the P. brasiliensis estrogen-binding protein. Because of biohazard problems in working with mycelial cultures of P. brasiliensis, all the hormone-binding studies to date had been performed on cytosol from yeast organisms; *
however, because we hypothesize that host estrogens act on the infective mycelial form, we believed it important to document the presence of an estrogen-binding protein in cytosol from mycelial-form organisms. Our experiments demonstrated that such a binding protein is present in mycelial-form P. brasiliensis. Moreover, competition studies revealed that diethylstilbestrol (DES), a nonsteroidal synthetic estrogen, has a higher affinity for the estrogenbinding protein in cytosol obtained from mycelial cultures than does cytosol obtained from yeast-form organisms. We also report new information on the estrogen-binding protein obtained from yeast cultures. These new data on the binding protein in cytosol from mycelial-form organisms indicate a greater concordance between hormone binding in vitro and bioactivity of hormones in culture than was previously indicated. We believe that these findings lend further credence to our hypothesis that P. brasiliensis possesses a functionally responsive, primitive hormone receptor system. MATERIALS AND METHODS Materials. Tritiated 17p-estradiol ([3H]estradiol) was purchased from the Amersham Corp., Arlington Heights, Ill. Radioinert steroids were purchased from Steraloids, Inc., Wilton, N.H. Tamoxifen was a gift from Stuart Pharmaceuticals, Wilmington, Del. All chemicals were reagent grade and were purchased from the Sigma Chemical Co., St. Louis, Mo., unless otherwise noted. Preparation of cytosol. Cytosol from yeast cultures was prepared as previously described (9). In brief, 300 ml of
Corresponding author. 199
200
STOVER ET AL.
INFECT. IMMUN. I
-I
0
12
It
I
0OC
-
I
I
~~~56°C
_
37OC~
I-
x
w
10
cc
Uz
81
0 co
6
-J
_
0 cc LU w
4 2
I
, I
0
500
1000 1500
JL 0
I
I
500
I
I
1000 1500
]I
.j
I0
500
1000 1500
[3H] ESTRADIOL BOUND (fmol/mI) FIG. 1. Scatchard analyses of [3H]estradiol binding in yeast-form P. brasiliensis cytosol pretreated at various temperatures. Cytosol pretreated at 0°C showed a single high-affinity binding site with an apparent dissociation constant (Kd) of 8.5 nM and an Nmax of 590 fmol of [3H]estradiol per mg of cytosol protein. Cytosol pretreated at 37°C showed no significant change in high-affinity binding but exhibited a new, low-affinity binding site. Cytosol pretreated at 56°C showed a loss of high-affinity binding and only low-affinity binding with a Kd of =150 nM.
McVeigh-Morton (MVM) liquid medium was inoculated with a 5-day-old culture from an MVM slant and incubated for 5 days at 35°C on a gyratory shaker at 150 rpm. Aliquots of 100 ml each were then added to 200 ml of fresh MVM medium and incubated for 10 days as described above. Contamination was excluded by subculturing cells on blood agar plates before harvesting. Cells were collected by centrifugation, washed twice in saline, and suspended in a homogenization medium containing 250 mM sucrose, 10 mM Tris, 1.5 mM EDTA, 12 mM monothioglycerol, 10 mM Na2MoO4, and 10% glycerol (pH 7.8). The cells were disrupted mechanically at 0°C by vigorous agitation with 250- to 300-,um glass beads. Cytosol was obtained by centrifugation of disrupted cells at 204,000 x g for 30 min. Mycelial cultures were grown by inoculating 200 ml of MVM medium plus 100 ml of yeast culture filtrate with small fragments of a 2- to 3-week-old MVM mycelial slant culture. Cultures were incubated at room temperature on a Gyrotory shaker (150 rpm) for 10 to 12 days. Mycelia were collected on filter paper, washed twice in saline and once in homogenization medium, and suspended in homogenization medium (50:50, vol/vol). Mycelial masses were then disrupted by six 15-s bursts with an electric grinding probe (Tissumizer; Tekmar Co., Cincinnati, Ohio), followed by vigorous agitation on a vortex mixer for 15 min with glass beads and cytosol preparation as described previously. Tubes were kept cold throughout the experiment by immersion in an ice slurry. Binding studies. Steroid-binding assays were performed as previously described (5). Briefly, 200-jil aliquots of cytosol were incubated with [3H]estradiol for 3 h at 0°C. Nonspecific binding of [3H]estradiol was assessed in all experiments by concurrent incubation of samples with a 500-fold molar excess of unlabeled estradiol. Specific binding of [3H]estradiol was calculated by subtracting nonspecific binding from total binding. Radioinert steroids were always added with 10-,u portions of 100% redistilled ethanol. Bound [3H]estradiol was separated from free [3H]estradiol by using a centrifuged gel-exclusion microcolumn of fine Sephadex G-50 (Pharmacia Fine Chemicals, Piscataway, N.J.) (5). Samples were loaded onto a column precentrifuged at 130 x
min. The column was recentrifuged for 3 min, and the eluate, containing protein-bound [3H]estradiol, was assayed for radioactivity. Cytosol protein concentration was measured by the Coomassie dye binding technique (1).
g for 2
RESULTS
High- and low-affinity [3H]estradiol-binding sites in yeast cytosol. In our previous studies with P. brasiliensis, we suggested that both high- and low-affinity binding sites for [3H]estradiol existed in cytosol from yeast cultures. We also reported that the [3H]estradiol binding appeared remarkably stable, even to heating to 56°C (5). The results of a representative study of the equilibrium binding characteristics of yeast-form P. brasiliensis binding sites are illustrated in Fig. 1. Cytosol was prepared as described before, and portions were incubated at 0, 37, or 56°C for 30 min. The cytosols
then chilled to 0°C and incubated with multiple concentrations of [3H]estradiol at 0°C for 3 h. Scatchard analysis (11) of the specific binding data obtained from cytosol incubated at 0°C showed that these data fit well to a straight line, suggesting the presence of a single class of noninteracting binding sites. This result was consistent with our previous findings (5, 9). The apparent dissociation constant (Kd) in this experiment was 8.5 nM, and the binding capacity (Nm,xe) was 1,028 fmol of [3H]estradiol per ml (590 fmol of [3H]estradiol per mg of protein). Similar analysis of the specific [3H]estradiol binding data from cytosol heated to 37°C revealed that the high-affinity binding site was present and stable at this temperature. However, the appearance of a second, lower-affinity [3H]estradiol-binding site could be detected (Fig. 1). This was in marked contrast to the single binding site detected in cytosol incubated at 0°C. When cytosol was preheated to 56°C, the binding data revealed only the presence of the lower-affinity [3H]estradiol-binding site, demonstrating the thermal lability of the high-affinity estrogen-binding protein. The Kd of the low-affinity site was estimated to be 150 nM, and the binding capacity, depending on the conditions employed, was estimated to be 3,000 to 4,500 fmol of [3H]estradiol per ml (1,700 to 2,600 fmol of [3H]estradiol per mg of protein). It should be noted that the measured affinity for estradiol were
VOL. 51, 1986
ESTRADIOL-BINDING PROTEINS FROM P. BRASILIENSIS
I
.(
iB)_
5 0.
201
m
a
01
0
0
w
x
E
4
E LL
cl z n
a
3
z
0
0
Do
-i
co
0
-
0
_
2
_~ ~ ~ ~ ~ ~-
\
a 4
cc
c-
1
wU
20
40
50
0
60
100
150
200
3H] ESTRADIOL BOUND [1-H1 ESTRADIOL (fmol/mg protein) CONCENTRATION (nM) FIG. 2. Equilibrium analysis of [3H]estradiol binding in cytosol prepared from mycelial-form P. brasiliensis. (A) Isotherm of total (0), specific (0), and nonspecific (A) binding at various [3H]estradiol concentrations. (B) Scatchard analysis of the specific binding data taken from the isotherm. The Kd in this study was 26 nM, and the binding capacity was 136 fmol of ['H]estradiol per mg of cytosol protein.
of the high-affinity site is approximately twice as high in this study as it was in a previous study (5). We now consistently observe Kd values of between 6 and 12 nM; we believe this difference is a result of improved technique, including the extreme care taken to ensure ice-cold conditions throughout the binding assay, thus minimizing interference from the low-affinity binding site, and the consistent use of high cytosol protein concentrations (initial concentration, -3 to 7 mg of protein per ml) to increase the total binding measured. [3H]estradiol-binding sites from mycelial cytosol. In preliminary experiments, we established that an estrogen-binding moiety was indeed present in mycelial-form P. brasiliensis by single-point analysis at high [3H]estradiol concentrations. Cytosols prepared from mycelial cultures of four clinical isolates of P. brasiliensis were incubated with 26 nM [3H]estradiol + a 500-fold excess of radioinert estradiol for 3 h at 0°C. These data indicate that in all four isolates, Ber (5), Gir (9), Ru (9), and Zan (new), specific binding of [3H]estradiol could be detected. These isolates bound 38, 308, 35, and 42 fmol of [3H]estradiol per mg of protein, respectively. All subsequent experiments were performed on the Ber isolate, since the estrogen-binding protein from yeast cultures of this isolate had been the one most extensively characterized (5). Scatchard analysis of [3H]estradiol binding to mycelial cytosol. The equilibrium binding characteristics of the mycelial estrogen-binding site were subsequently examined by incubation of cytosol with multiple concentrations (6.5 to 65 nM) of [3H]estradiol at 0°C for 3 h. The results of one such study are shown in Fig. 2. The isotherm shows total, nonspecific, and specific binding as a function of [3H]estradiol concentration (Fig. 2A). Specific binding was saturable, and nonspecific binding was linear and ranged from 20 to 45% of the total binding. When the specific binding data are plotted by the method of Scatchard (11), the data points fit well to a straight line. The mean values of three such experiments were 13 nM for the Kd (range, 6 to 26 nM) and 78 fmol of [3H]estradiol per mg of protein for the
binding capacity (range, 45 to 136 fmol of [3H]estradiol per mg of protein). Displacement analysis of [3H]estradiol binding to mycelial cytosol. Additional binding studies were performed to assess the specificity of the mycelial binding site for [3H]estradiol. Radioinert competitors were selected based on previous results from both binding specificity studies and transformation experiments. Specifically, we tested the capacity of unlabeled estradiol, DES, and tamoxifen to displace [3H]estradiol from the estrogen-binding protein in cytosol from mycelial cultures. The pooled results of several such studies are presented in Fig. 3. Unlabeled 17,-estradiol, as expected, was the most potent competitor, achieving 50%
I 1
z O
I
I
I'
1
I
I
I
I
I
I.
100 TAM
-
0 5
_
75
-
0*-f -
40c 50
0 0Q. _ cc-,
