Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

CURRENT MICROBIOLOGY Vol. 37 (1998), pp. 141–143

An International Journal

R Springer-Verlag New York Inc. 1998

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis Gioconda San-Blas, Franc¸oise Sorais, Gustavo Nin˜o-Vega, Cioly Me´ndez, Felipe San-Blas Instituto Venezolano de Investigaciones Cientificas (IVIC), Centro de Microbiologı´a y Biologı´a Celular, Apartado 21827, Caracas 1020A, Venezuela Received: 29 September 1997 / Accepted: 19 February 1998

Abstract. Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH’s between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process.

Many fungal pathogens, such as Candida albicans, Paracoccidioides brasiliensis, and Coccidioides immitis, produce extracellular or cell wall-bound proteinases that may play a role in pathogenicity [9], endosporulation [6], or antigenicity [5]. These enzymes are mainly aspartic [19], or serine proteinases [3, 20]. Cytosolic proteinases, on the other hand, may serve as precursors of their extracellular form [8], or they are involved in metabolic processes related to morphogenesis, such as the synthesis of cell wall glucan [7] and chitin [15]. Hence, we screened cytosolic proteinases from P. brasiliensis, a dimorphic fungus known to cause a frequently encountered systemic mycosis in Latin America. P. brasiliensis strain IVIC Pb73 (ATCC 32071) is kept and grown as previously reported [16]. Cytosolic fractions from Y cells and M cultures and from cultures in phase transition were prepared as before [17]. Proteinases in cytosol (100–200 µg protein per tube) were assayed from 23° to 55°C, with azocoll [4] as substrate. One

Correspondence to: G. San-Blas

proteolytic unit (PU) was the amount of protein needed to increment 0.1 unit of absorbance at 520 nm in 1 h. Specific proteolytic units (SPU) were PU/mg protein. Molecular-sieve chromatography was performed in a Sephacryl S-200 column (90 cm 3 2.5 cm), with 10 mM Tris-HCl buffer, pH 7.0. Ion-exchange chromatography was done in a DEAE-cellulose 32 (Whatman; 12 cm 3 1.5 cm) column equilibrated with 10 mM Tris-HCl buffer, pH 7.0, and eluted with a stepwise gradient (100 ml initial buffer followed by 125 ml of 0.3 M NaCl in buffer). SDS-PAGE electrophoresis [10] and gelatin zymograms [13] were also run. Tests for proteinase inhibitors were done in the presence of the following: PMSF, tosyl lysine chloromethyl ketone (TLCK), antipain, soybean trypsin inhibitor (SBTI), chymostatin, EDTA, 1,10-phenanthroline, and iodoacetamide, at concentrations of 0.04 and 0.4 mM (SBTI and chymostatin used at 0.4 and 4 µg/ml). Reagents were from Sigma Chemical Co. (St. Louis, Mo). Cytosolic proteinases were able to digest azocoll, azocasein and hemoglobin within the pH range 6.0–9.0. Optimal temperature was 45°C. Activity was always higher in mycelial preparations as compared with yeasts,

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Table 1. Effect of inhibitors on the cytosolic proteinases from P. brasiliensis, Y and M phases % inhibition of activity over control Inhibitor PMSF TLCK Antipain EDTA 1,10-Phenanthroline I-acetamide

SBTI Chymostatin

Phase

0.04 mM

0.4 mM

Y M Y M Y M Y M Y M Y M

96.2 100.0 0 0 100.0 100.0 0 0 0 5.9 0 34.3

96.6 100.0 0 0 100.0 100.0 0 44.0 0 6.7 0 37.2

0.4 µg/ml

4 µg/ml

0 58.0 100.0 100.0

0 59.1 100.0 100.0

Y M Y M

Assays were performed in duplicate, and mean values are reported.

and 10 times higher at 37°C [SPU 129 (Y) and 297 (M)] than at 23°C [SPU 16 (Y) and 27 (M)]. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelium (SPU 20, 41, and 58 at 4, 7, and 24 h in transition, respectively). The mycelium to yeast transition involved a decrease in proteinase activity (SPU 48, 25, 22 at the same times). Elution of the cytosolic fraction through Sephacryl S-200 produced a broad peak with maximal activities at 54 and 78 kDa for the M and Y preparations, and protein purifications of 8.1 and 8.9 times, respectively. Ionexchange chromatography concentrated enzymatic activities at an ionic strength of 0.1 M NaCl, with purifications of 40.7 and 32.7 times, for the Y and M phases. Inhibitors of serine proteinase such as antipain, PMSF, or chymostatin, but not TLCK or SBTI, were 100% efficient in inhibiting both Y and M proteinase activities (Table 1). Iodoacetamide, an inhibitor of cysteine proteinases, affected partially (34–37%) the activity of the M enzymatic preparation but not that of the Y proteinase. The behavior was similar for 1,10-phenanthroline and EDTA, inhibitors of metallo proteinases, with 7% and 44% inhibitions, respectively. SDS-PAGE electrophoresis produced one main band of molecular weight 50 kDa in the Y phase (Fig. 1A). In the M phase, instead, only a series of weak bands

were seen. In gelatin zymograms (Fig. 1B), the M proteinases resolved into three strong bands (molecular weights 58, 92, and 112 kDa), additional weaker bands at 128 and 225 kDa, and a blurred zone between 60 and 84 kDa. Instead, the Y proteinase appeared as a single band around 70 kDa, plus a diffuse and broad zone between 84 and 90 kDa. In P. brasiliensis, extracellular collagenase, elastase, and gelatinase have been reported [2, 18]. Carmona and colleagues [3] also found an exocellular serine-thiol proteinase with a very restrictive substrate specificity, and major activity at basic pH. The cytosolic enzymes reported here may classify as serine proteinases (EC 3.4.21), according to their strong inhibition by PMSF, chymostatin, and antipain. However, they differ in some respects. The single band of proteinase activity in the Y phase was not affected by EDTA, iodoacetamide, 1,10phenanthroline, or SBTI. Hence, it is not a metallo- or a cysteine proteinase. But it may have a chymotrypsinrather than a trypsin-like nature like those reported in the cytoplasm of Geotrichum candidum [12] and T. rubrum [11]. The several bands in mycelial preparations also have characteristics of serine proteinases, with additional activities of cysteine- and metallo-proteinases that were partially inhibited by EDTA, 1,10-phenanthroline, and iodoacetamide. Such proteinase diversity is also found in other fungi, for example, A. nidulans, whose intracellular serine proteinases (I and II) are inhibited by PMSF but not by TLCK, TPCK, or benzamidine, differing markedly from trypsin and chymotrypsin [1]. Molecular weights of P. brasiliensis intracellular proteinases were higher than those from A. nidulans (30 and 31 kDa) [1], T. rubrum (34–37 kDa) [11], or Allomyces arbuscula (43 kDa) [14], but lower than that of G. candidum (278 kDa) [12]. A relationship between germination and proteolytic activity was recently suggested for A. arbuscula [14]. In P. brasiliensis, proteinase activity increased in preparations obtained from cells undergoing Y = M transition, and decreased in the reverse process, if the activity was measured at 37°C in vitro (at 23°C, proteinase activity was always very low). This may suggest that the yeast phase, but not the hyphal cells, would be able to use cytosolic proteinases in an effective way, or that mycelia have a pool of proteinases to be preferentially used in transforming to the yeast phase, once the temperature is raised to 37°C. Whether this result is meaningful to the dimorphic process remains to be analyzed with the help of dimorphic mutants.

G. San-Blas et al.: P. brasiliensis Proteinases

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Fig. 1. P. brasiliensis cytosolic preparation ex-DEAE cellulose. (a) SDS-PAGE electrophoresis; (b) Gelatin zymograms. Molecular weight of standards (kDa) to the left; those of samples, to the right.

ACKNOWLEDGMENTS This work has been supported by Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICIT) (grants S1-96000156 and PI960001292) and International Centre for Genetics and Experimental Biology (ICGEB) [grant CRP/VEN95-01(h1)]. Thanks to Dr. Veronica Hearn for kindly revising the manuscript.

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