Chymotrypsin- and trypsin-like activities secreted by the multidrug-resistant yeasts forming the Candida haemulonii complex

Abstract

Abstract:Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (K_m) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.

Key words
Brazilian clinical isolates; Candida haemulonii complex; chymotrypsin; serine peptidases; trypsin; virulence factors

INTRODUCTION

Candidiasis is one of the most frequent nosocomial infections worldwide and has drawn attention, in recent years, due to its significant contribution to the increased mortality rate of immunocompromised/immunosuppressed patients (Lockhart 2014, Agarwal et al. 2017). Although Candida albicans remains the most commonly isolated fungal species in hospital settings, infections by non-albicans Candida species have been increasingly reported over the last decades in a global scale (Agarwal et al. 2017).

The Candida haemulonii complex is composed by three phenotypically indistinguishable fungal species: C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera. Relevantly, these emerging yeasts are intrinsically resistant to the main antifungals commonly used in the clinical arena, including amphotericin B and different azoles (e.g., fluconazole, itraconazole and voriconazole) as well as they present limited susceptibility to echinocandins. Consequently, this multidrug-resistant profile has hampered the treatment of patients with systemic infections and increased the number of clinical failures and related cases of death caused by these fungal species (Rodero et al. 2002, Khan et al. 2007, Kim et al. 2009, Ruan et al. 2010, Crouzet et al. 2011, Cendejas-Bueno et al. 2012, Li et al. 2015, Ramos et al. 2015, Fang et al. 2016). Although C. haemulonii species complex infections have gained prominence in recent years, the interactions of these yeasts with the host remain poorly studied. In order to change this scenario, our research group has been engaged since 2015 in characterizing several aspects of the physiology and virulence of these emerging pathogens, such as antifungal susceptibility, biofilm formation and secretion of hydrolytic enzymes (Ramos et al. 2015, 2016, 2017). In this context, Ramos et al. (2016) described the production of aspartic-type peptidase in 12 Brazilian clinical isolates of C. haemulonii species complex, as judged by the degradation of albumin molecules incorporated into agar plates, in which C. haemulonii and C. haemulonii var. vulnera were classified as good producers and C. duobushaemulonii as excellent producers. In addition, caseinolytic activity was detected in all tested isolates, suggesting the secretion of other peptidase classes by this fungal complex.

Historically, several papers have pointed out to the relevance of secreted serine peptidases as virulence factors in fungi (Muszewska et al. 2017). In this sense, serine peptidases are involved in different contexts of fungal-host interplays, and they are important for nutrient acquisition from protein-rich sources (Reddy et al. 1996, Muszewska et al. 2017). In Candida spp., serine peptidases presenting hydrolytic activities in a wide range of pH as well as with the ability to hydrolyze key host proteins, such as components of the extracellular matrix and serum proteins, have already been described in C. albicans, C. guilliermondii, C. parapsilosis, C. tropicalis, C. dubliniensis and C. lipolytica (Santos and Soares 2005, Santos et al. 2006, Melo et al. 2007, Vermelho et al. 2010, Portela et al. 2010). Secreted serine-type peptidases belonging to the S1 family (e.g., trypsin-, chymotrypsin- and elastase-like peptidases) (Dubovenko et al. 2010) have gained prominence due to their multiple roles in the pathogenicity of opportunistic fungi (Muszewska et al. 2017), such as Scedosporium, Sporothrix and Aspergillus (Muhsin et al. 1997, Barata et al. 2002, Han et al. 2017). These serine peptidases have great similarities in their sequences and tridimensional structures; however, they present different substrate specificities (Haën et al. 1975, Ma et al. 2005), in which trypsin-like peptidases have affinity for basic amino acid residues (e.g., lysine and arginine), chymotrypsin-like peptidases have affinity for aromatic amino acid residues (e.g., phenylalanine, tyrosine and tryptophan) (Vajda and Szabó 1976), while elastase-like peptidases have preference for aliphatic amino acid residues, particularly alanine (Zimmerman and Ashe 1977).

So far, almost nothing is known about the secretion of serine-type peptidases by C. haemulonii species complex (Ramos et al. 2016, Souto et al. 2019). In this context and bearing in mind the importance of the S1 family of serine peptidases in many aspects of the fungus-host interaction, in the present study we have described the presence of serine peptidases in clinical isolates of C. haemulonii, C. haemulonii var. vulnera and C. duosbushaemulonii.

MATERIALS AND METHODS

CHEMICALS

The chromogenic peptide substrates N-succinyl-Ala-Ala-Pro-Phe-pNa (chymotrypsin detection), N-succinyl-Ala-Ala-Pro-Leu-pNa, N-succinyl-Ala-Ala-Ala-pNa (elastase detection), N-benzoyl-Phe-Val-Arg-pNa and Nα-benzoyl-DL-4Arg-pNa (trypsin detection), the peptidase inhibitors phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), Nα-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), benzamidine, 1,10-phenanthroline, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) and pepstatin A, as well as bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Buffer components were purchased from Isofar (Duque de Caxias, RJ, Brazil). Culture medium components were provided by Neogen (Lansing, MI, USA). All other reagents were of analytical grade.

MICROORGANISMS AND GROWTH CONDITIONS

Three Brazilian clinical isolates of the C. haemulonii complex were used in all the experiments performed in the present study: C. haemulonii (LIPCh7 from toenail), C. haemulonii var. vulnera (LIPCh9 from urine) and C. duobushaemulonii (LIPCh10 from bronchoalveolar lavage) (Ramos et al. 2015). Fungi were cultured in Sabouraud-dextrose medium (400 ml) at 37°C for 48 h under constant agitation (200 rpm). The clinical fungal isolates studied herein have reached their exponential phase at 48 h of in vitro growth on Sabouraud-dextrose liquid medium as previously published by our research group (Ramos et al. 2017).

COLLECTION OF THE CULTURE SUPERNATANTS

The fungal cultures were harvested by centrifugation (4.000×g, 10 min, 4°C) and the supernatants were filtered in a 0.22-µm membrane (Millipore, São Paulo, Brazil). The cell-free culture supernatants were concentrated 100-fold in an Amicon® ultrafiltration system (Amicon®, Beverly, USA) using a 10-kDa cut-off membrane (Santos and Soares 2005). The same volume of Sabouraud medium was also concentrated and used as a control to check for possible peptidase activity, showing negative results. Protein concentration of the culture supernatants was determined by the method described by Lowry et al. (1951), using BSA as standard.

CHARACTERIZATION OF EXTRACELLULAR SERINE PEPTIDASE ACTIVITY

The extracellular serine peptidase activities were determined, initially, in 10 mM sodium phosphate buffer (pH 7.0) using pNA-labeled peptide substrates for chymotrypsin, elastase and trypsin. Then, substrates displaying the highest hydrolysis for serine peptidase activities were selected for further studies as described below.

Modulation of serine peptidase activity by different pHs and temperatures was evaluated, respectively, using 0.1 M sodium citrate (pH 5.0), 0.1 M citrate phosphate (pH 6.0), 10 mM sodium phosphate (pH 7.0), 0.1 M Tris-HCl (pH 8.0), 0.1 M sodium glycine-hydroxide (pH 9.0-10.0) and 50 mM sodium phosphate (pH 11.0) as buffer solutions, and by incubating the supernatants at 25, 37, 45 or 55°C. For this last analysis, the pH of higher activity for each selected substrate was used. In addition, all other assays were performed at 37°C. The effects of proteolytic inhibitors on peptidase activities were evaluated after incubation with PMSF (0.0001-1 mM), AEBSF (0.1-10 mM), benzamidine (1-10 mM), 1,10-phenanthroline (1-10 mM), TPCK (0.01-0.2 mM), TLCK (0.01-0.2 mM), E-64 (0.01 mM) or pepstatin A (0.01 mM). Systems with no proteolytic inhibitor were used as controls. The enzymatic activities assayed in the absence of inhibitors was considered as 100%. Kinetic parameters were determined by incubating the culture supernatants with increasing concentrations of the substrates for which higher serine peptidase activities were detected. The apparent K_m and V_max for these substrates were determined by non-linear regression analysis applying Michaelis–Menten kinetics using the Prism v5.03 program (GraphPad Software, Inc.). For all assays, supernatants to a final concentration of 0.5 μg/µl total extracellular proteins were added to different substrates (final concentration ranging from 0.1 to 1 mM for kinetic parameters, and 100 µM for further analysis) in the desired buffer solutions. The release of pNa was evaluated in 96-well plates by spectrophotometry at 410 nm during 60 min of incubation at 5-min intervals between each reading. The specific serine peptidase activity was expressed as nmol of pNa released per min and mg of total protein in concentrated culture supernatants. The concentration of pNa was calculated using a molecular extinction coefficient of 8270 M-¹.cm-¹. The effect of inhibitors was expressed as percentage of residual peptidase activity in relation to untreated controls.

STATISTICAL ANALYSIS

All the experiments were performed in triplicate, in three independent experimental sets. Data were expressed as mean ± standard deviation. The results were evaluated by analysis of variance (ANOVA) followed by Tukey’s test using the Prism v5.03 program (GraphPad Software, Inc.). In all analyses, P values ≤0.05 were considered statistically significant.

RESULTS

SECRETION OF SERINE PEPTIDASES BELONGING TO THE S1 FAMILY

Initially, secretion of serine peptidases belonging to the S1 family was evaluated in the culture supernatants from three clinical isolates of the C. haemulonii complex employing five distinct substrates to detect chymotrypsin-, trypsin- and elastase-like activities. These three types of enzymatic classes were detected in all supernatants (Fig. 1). Interestingly, no activity was detected against the Nα-benzoyl-DL-4Arg-pNa substrate, typical for trypsin. With respect to the other substrates, higher serine peptidase activities were detected, after 60 min, in the supernatant of C. haemulonii followed by C. duobushaemulonii and C. haemulonii var. vulnera (Fig. 1, insert).

Figure 1
Serine peptidase activities measured in the culture supernatants of clinical isolates of the C. haemulonii complex. The serine peptidase activities were evaluated during 60 min of incubation at pH 7.0 and 37°C. Five substrates were used: N-succinyl-Ala-Ala-Pro-Phe-pNa (S1) for chymotrypsin, N-succinyl Ala-Ala-Pro-Leu-pNa (S2) and N-succinyl-Ala-Ala-Ala-pNa (S3) for elastase, and N-benzoyl-Phe-Val-Arg-pNa (S4) and Nα-benzoyl-DL-4Arg-pNa (S5) for trypsin. The inserts show the average of serine peptidase activity against each substrate at 60 min. Values represent means ± SD from at least three independent experiments.

BIOCHEMICAL PROPERTIES OF CHYMOTRYPSIN- AND TRYPSIN-LIKE PEPTIDASES

Since higher activities were detected over N-succinyl-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, these substrates were selected to investigate the biochemical properties of chymotrypsin- and trypsin-like peptidases secreted by C. haemulonii species complex, respectively.

EFFECT OF pH AND TEMPERATURE

In general, serine peptidase activities were detected in a wide pH range (varying from 5.0 to 11.0) (Fig. 2). However, for all analyzed samples, pHs 7.0 and 9.0 were those of greater chymotrypsin- and trypsin-like activities, respectively. Besides that, these hydrolytic activities were higher at temperatures of 37-45°C, although activities were also detected at 25 and 55°C (Fig. 3).

Figure 2
Effect of pH on chymotrypsin- and trypsin-like activities measured in the culture supernatants of clinical isolates of the C. haemulonii complex. The serine peptidase activities were evaluated by spectrophotometry in a pH range varying from 5.0 to 11.0 for 60 min at 37°C. Two substrates were used: N-succinyl-Ala-Ala-Pro-Phe-pNa for chymotrypsin and N-benzoyl-Phe-Val-Arg-pNa for trypsin. The bars represent the mean ± SD from at least three independent experiments.

Figure 3
Effect of temperature on chymotrypsin- and trypsin-like activities measured in the culture supernatants of clinical isolates of the C. haemulonii complex. The serine peptidase activities were evaluated by spectrophotometry for 60 min at 25, 37, 45 and 55°C. Two substrates were used: N-succinyl-Ala-Ala-Pro-Phe-pNa for chymotrypsin and N-benzoyl-Phe-Val-Arg-pNa for trypsin, at pH 9.0. The bars represent the mean ± SD from at least three independent experiments.

EFFECT OF PEPTIDASE INHIBITORS

The effect of inhibitors against serine peptidase activities are shown in Table I. Chymotrypsin-like activities detected in supernatants of all fungal isolates were inhibited by AEBSF and PMSF, in a concentration-dependent manner. At higher concentrations, AEBSF (10 mM) and PMSF (0.1 mM) reduced the enzymatic activities up to 80 and 100%, respectively. Besides that, TPCK (0.2 mM) also induced a slight reduction (≈18%) on these peptidase activities. In a similar way, trypsin-like activities detected in all evaluated supernatants were also inhibited by AEBSF and PMSF in a typically concentration-dependent fashion. Additionally, the inhibition of these trypsin-like activities by approximately 100, 75 and 40% was measured in the presence of 10 mM AEBSF, 1 mM PMSF and 10 mM benzamidine, respectively. Differently, TLCK, 1,10-phenanthroline, E-64 and pepstatin A did not affect the hydrolytic activities of both chymotrypsin- and trypsin-like peptidases.

TABLE I
Effect of peptidase inhibitors on the serine peptidase activities released by C. haemulonii species complex.93.4 ± 4.7

		Residual peptidase activity (%)
		Chymotrypsin-like				Trypsin-like
Inhibitor (I)	I [mM]	C. haemulonii	C. haemulonii var. vulnera	C. duobushaemulonii	C. haemulonii	C. haemulonii var. vulnera	C. dubushaemulonii
Control	0	100.0	100.0	100.0	100.0	100.0	100.0
PMSF	10-4	102.0 ± 0.2	100.3 ± 5.2	102.9 ± 1.1	-	-	-
	10-3	88.2 ± 4.0	89.7± 8.4	87.6 ± 0.8	-	-	-
	10-2	26.8 ± 2.0*	32.6 ± 4.5*	32.0 ± 3.0*	99.1 ± 1.1	92.5 ± 7.3	100.9 ± 1.5
	10-1	1.4 ± 0.3*	2.7 ± 0.4*	0.80 ± 0.1*	93.7 ± 5.5	84.0 ± 2.6*	91.3 ± 6.1
	1	-	-	-	22.4 ± 1.5*	38.2 ± 3.8*	24.4 ± 5.0*
AEBSF	10-1	95.9 ± 4.2	82.6 ± 1.3*	98.0 ± 0.7	98.1 ± 0.4	90.9 ± 3.6	88.0 ± 6.5
	1	84.1 ± 5.6*	66.0 ± 1.8*	70.4 ± 2.1*	64.0 ± 1.9*	64.2 ± 2.5*	89.5 ± 2.2
	10	24.9 ± 3.3*	17.8 ± 1.1*	34.0 ± 3.6*	2.4 ± 0.3*	1.5 ± 0.1*	7.9 ± 0.2*
Benzamidine	1	98.7 ± 5.3	100.6 ± 5.6	104.1 ± 5.0	98.4 ± 4.9	91.29 ± 3.0	95.6 ± 2.2
	5	99.4 ± 7.2	93.1 ± 3.8	85.0 ± 1.1	88.3 ± 1.1	70.5 ± 2.0*	86.7 ± 3.5
	10	101.0 ± 5.2	92.3 ± 1.8	83.0 ± 4.0	75.1 ± 0.4*	60.3 ± 2.4*	60.0 ± 2.1*
TPCK	10-2	92.4 ± 3.6	103.7 ± 5.3	101.9 ± 2.7	98.95 ± 1.1	96.5 ± 3.4	102.6 ± 3.7
	10-1	85.7 ± 4.7*	97.4 ± 6.7	87.5 ± 2.2	95.7 ± 0.9	100.1 ± 3.5	98.6 ± 3.2
	2x10-1	83.8 ± 1.6*	81.4 ± 5.2*	85.0 ± 1.3*	98.9 ± 5.3	93.2 ± 2.0	95.7 ± 5.9
TLCK	10-2	97.0 ± 4.5	106.2 ± 7.6	102.3 ± 3.5	101.8 ± 5.5	96.1 ± 4.5	96.6 ± 6.56
	10-1	96.9 ± 4.5	104.4 ± 4.5	104.1 ± 4.7	100.7 ± 5.2	99.3 ± 1.5	95.9 ± 6.1
	2x10-1	94.2 ± 5.7	95.0 ± 6.4	101.4 ± 4.3	100.0 ± 6.2	94.4 ± 6.8	96.5 ± 6.2
1,10-Phenanthroline	1	93.4 ± 4.7	99.0 ± 2.6	101.5 ± 2.1	94.8 ± 5.2	100.4 ± 2.9	91.6 ± 6.4
Pepstatin	10-2	100.4 ± 0.1	101.4 ± 4.2	101.1 ± 0.8	100.2 ± 0.6	98.8 ± 3.7	101.1 ± 4.8
E-64	10-2	104.6 ± 6.3	99.3 ± 4.7	100.3 ± 1.1	106.1 ± 5.7	99.1 ± 3.8	99.1 ± 6.1

• The culture supernatants were incubated with different concentrations of peptidase inhibitors at 37°C for 60 min and the enzyme activity measured in the absence of any inhibitors was taken as 100% (Control). Residual chymotrypsin- and trypsin-like activities were detected in the supernatants from degradation of the N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa italicstrates, respectively. Data reflect the mean ± SD from at least three independent experiments; *significant difference in relation to the control group (P < 0.05).

KINETIC PARAMETERS

The kinetic parameters of the chymotrypsin- and trypsin-like peptidases detected in the C. haemulonii, C. hameulonii var. vulnera and C. duobushaemulonii secretions were also evaluated (Table II). In general, apparent K_m and V_max values for N-succinyl-Ala-Ala-Pro-Phe-pNa ranged from 0.96 to 0.99 mM and 1.09 to 7.0 nmol pNA.mg-¹.min-¹, respectively. In turn, for N-benzoyl-Phe-Val-Arg-pNa, apparent K_m ranged from 0.098 to 0.17 mM and V_max from 1.0 to 1.87 nmol pNA.mg-¹.min-¹. By one-way ANOVA test, no significant difference was observed between the apparent K_m values found for the chymotrypsin-like serine peptidases. On the other hand, the apparent K_m value found for the trypsin-like serine peptidase produced by C. haemulonii differed significantly (P<0.05) from those found in C. haemulonii var. vulnera and C. duobushaemulonii.

TABLE II
Kinetic parameters of the extracellular serine peptidase activities released by C. haemulonii species complex.

Species	Serine-type peptidase	Km (mM)	Vmax (nmol pNA.mg-1.min-1)
C. haemulonii	Chymotrypsin-like	0.96 ± 0.12	7.00 ± 0.53
	Trypsin-like	0.10 ± 0.01	1.87 ± 0.08
C. haemulonii var. vulnera	Chymotrypsin-like	0.98 ± 0.20	1.09 ± 0.14
	Trypsin-like	0.14 ± 0.02	1.00 ± 0.04
C. duobushaemulonii	Chymotrypsin-like	0.99 ± 0.09	4.52 ± 0.27
	Trypsin-like	0.17 ± 0.02	1.71 ± 0.08

DISCUSSION

In recent years, the rapid emergence of multidrug resistant non-albicans Candida species, such as those belonging to the C. haemulonii complex, has stimulated the search for new therapeutic agents and specifically serine peptidase inhibitors have emerged as a valuable alternative to test their antifungal potential (Santos and Soares 2005, Santos et al. 2006, Melo et al. 2007, Vermelho et al. 2010, Portela et al. 2010, Santos 2011, Whaley et al. 2016). Among the secreted serine peptidases correlated with the fungal pathogenesis in humans, we can highlight those belonging to the S1 family (one of the main families of fungal serine peptidases), which are well described in several genera of filamentous fungi, but very little studied in Candida spp. (Muhsin et al. 1997, Barata et al. 2002, Zarnowski et al. 2007, Dubovenko et al. 2010, Muszewska et al. 2017). In the literature, several studies have proposed that serine peptidases belonging to the S1 family play many pathophysiological roles, acting as virulence factors in different pathogenic fungi. In Sporothrix schenckii, for example, chymotrypsin-like serine peptidases enable the fungus to invade the skin and to obtain peptides from insoluble protein sources during the interaction with the host (Tsuboi et al. 1987). In the same way, secreted elastase-like serine peptidases from Aspergillus fumigatus assist this pathogen in lung infection during invasive arpergillosis (Kolattukudy et al. 1993). In turn, the secretion of trypsin-like peptidases has already been described in opportunistic pathogens such as Scedosporium aurantiacum (Han et al. 2017). Herein, the secretion of serine peptidases from the S1 family, including chymotrypsin-, trypsin- and elastase-like activities, was reported in C. haemulonii species complex.

Peptidases of microorganisms present unique characteristics with respect to the catalytic mechanism, substrate specificity, and optimal temperature and pH (Rao et al. 1998). Since peptidases participate in different aspects of the fungus-host interaction, their biochemical characterization is interesting not only for the understanding of the enzyme as a whole, but also for the knowledge of its possible functions during the infection with the objective of developing new therapeutic strategies. In view of this, secreted trypsin- and chymotrypsin-like serine peptidases, which provided the greatest activities in the clinical isolates used in the present study, were evaluated in relation to the influence of pHs, temperatures and peptidase inhibitors on the enzyme activities, as well as regarding its kinetic parameters. Extracellular chymotrypsin- and trypsin-like serine peptidases exhibited activities in a wide range of pHs and temperatures. Since Candida spp. is exposed to a pH range from slightly alkaline to acidic (Davis 2009) and to possible temperature changes (Robert et al. 2015) during infection of the human host, the secretion of peptidases that adapt well to such variations probably will favor their survival, colonization and growth. In parallel, inhibition of both activities by PMSF and AEBSF confirmed the class of these enzymes as being serine peptidases. Besides that, the reduction of the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa by TPCK and benzamidine, respectively, confirmed that these enzymes are chymotrypsin- and trypsin-like serine peptidases, since they are typical targets of such inhibitors. In agreement with these data, no inhibition was observed for pepstatin A, E-64 and 1,10-phenanthroline (inhibitors of aspartic-, cysteine- and metallopeptidases, respectively). Interestingly, TLCK, known to primarily inhibit trypsin-like serine peptidases, did not affect the activities of these enzymes. Secreted serine peptidases from the S1 family have been described mainly in human pathogenic, phytopathogenic and entomopathogenic fungi. In accordance with the results obtained here, a secreted chymotrypsin-like serine peptidase able to degrade the substrate N-succinyl-Ala-Ala-Pro-Phe-pNa, with higher activity at pH 7.0 and inhibited by PMSF, has been described in the entomopathogenic fungus Cordyceps sinensis (Zhang et al. 2008). A secreted trypsin-like serine peptidase able to degrade the substrate N-benzoyl-Phe-Val-Arg-pNa, with higher activity at pH 8.5 and inhibited by PMSF, was described in the phytopathogenic fungi Stagonospora nodorum (Carlile et al. 2000). With regard to human pathogenic fungi, secretion of chymotrypsin-like serine peptidases able to degrade the substrate N-succinyl-Ala-Ala-Pro-Phe-MCA and whose activity is inhibited by PMSF, was detected in the culture supernatant of the fungus S. schenckii (Tsuboi et al. 1987). In parallel, a secreted trypsin-like peptidase, with higher activity in basic pH at 45°C and inhibited by benzamidine, was described in the culture supernatant of Fusarium oxysporum (Barata et al. 2002).

In the present study, secreted serine peptidases from the S1 family were described, for the first time, in the C. haemulonii species complex, opportunistic fungal pathogens with extremely scarce knowledge about their pathophysiology processes. The broad range of pH and temperature at which these enzymes have hydrolytic activity suggests their abilities to function in several sites of human body and natural environments, presenting different physicochemical conditions. However, more accurate analyzes will be necessary to better investigate this hypothesis and the functions of these enzymes in cell biology and virulence of these fungi.

ACKNOWLEGMENTS

This study was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001).

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Publication Dates

• Publication in this collection
  23 Sept 2019
• Date of issue
  2019

History

• Received
  23 July 2018
• Accepted
  18 Oct 2018