Secreted proteases from pathogenic fungi

Int. J. Med. Microbiol. 292, 405 ± 419 (2002) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/ijmm

Secreted proteases from pathogenic fungi Michel Monoda , Sabrina Capocciaa, Barbara Le¬chennea, Christophe Zaugga, Mary Holdomb, Olivier Joussona a b

Service de Dermatologie (DHURDV), Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Department of Dermatology, St John`s Institute, Guy`s Hospital, London, UK

Abstract Many species of human pathogenic fungi secrete proteases in vitro or during the infection process. Secreted endoproteases belong to the aspartic proteases of the pepsin family, serine proteases of the subtilisin family, and metalloproteases of two different families. To these proteases has to be added the non-pepsin-type aspartic protease from Aspergillus niger and a unique chymotrypsin-like protease from Coccidioides immitis. Pathogenic fungi also secrete aminopeptidases, carboxypeptidases and dipeptidyl-peptidases. The function of fungal secreted proteases and their importance in infections vary. It is evident that secreted proteases are important for the virulence of dermatophytes since these fungi grow exclusively in the stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. For Aspergillus fumigatus, the role of proteolytic activity has not yet been proved. Although the secreted proteases have been intensively investigated as potential virulence factors, knowledge on protease substrate specificities is rather poor and few studies have focused on the research of inhibitors. Knowledge of substrate specificities will increase our understanding about the action of each protease secreted by pathogenic fungi and will help to determine their contribution to virulence. Key words: proteases ± virulence ± Candida ± Aspergillus ± dermatophytes ± adherence

Introduction More than 100,000 species of fungi are described, but only about 400 are known to be human pathogens or to cause opportunistic infections in neutropenic patients (de Hoog and Guarro, 1995). Among various potential virulence factors proposed, secreted proteolytic activity has been intensively investigated, and the presence of different secreted proteases has been demonstrated on the surface of fungal elements colonizing mucosa and penetrating tissues during disseminated infections.

The aim of this paper is to briefly review different properties of the proteases secreted by pathogenic fungi and to discuss their role as virulence factors.

Classification of proteases The term protease is synonymous with peptidase, proteolytic enzyme and peptide hydrolase. The proteases include all enzymes that catalyse the cleavage of the peptide bonds (CO NH) of proteins, digesting these proteins into peptides or free

Corresponding author: Michel Monod, Service de Dermatologie, Laboratoire de Mycologie, BT422, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland. Phone: ‡ 41 21 314 0376, Fax: ‡ 41 21 314 0378, E-mail: [email protected]

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Clan

Endoproteases AA A1

Candida albicans

Candida tropicalis

Candida Rhizopus Aspergillus § Aspergillus parapsilosis spp. fumigatus flavus/oryzae

Aspergillus niger

Sap1-Sap6 A01.014

Sapt4, A01.014

Sapp1, Sapp2 A01.038

PepA A01.016

Sap8 A0.037 Sap7 unassigned

Sapt1 A0.037 Sapt2, Sapt3, unassigned

Protein family A01.012

Pep1 A01.026

PepO A01.026

Dermatophytes

Coccidioides immitis

Pep4 ˆ PrA A01.018

Pep2 A01.025

Sap9, Sap10 unassigned

Yapsins A01.030

AX

A4

MA( M )

M35

ß1

MA( E )

M36

SB

S8A

Mep M36.001 Alp1 S08.053 Alp2 S08.052

SX

S19

Exoproteases SC S9B S9C MH

M28B

SC

S10

Saccharomyces cerevisiae

PepB A04.002. NpII M35.002 NpI M36.001 Alp S08.053

Protein family PepD S08.053

Protein family

ProtB S08.052 Chymotrypsinlike protease S19.001

DppIV S09.008 DppV S09.012

DppIV S09.008 DppV S09.012 Aminopeptidase (60 kDa) unassigned Aminopeptidase (35 kDa) M28.006 Caboxypeptidase (60 kDa) unassigned

DppIV ß2 DppV S09.012

CPDI-S10.014 CPDII ˆ PepF S10.006

DppB S09.006 DppA ˆ Ste13 S09.005 Aminopeptidase Y M28.001

Carboxypeptidase Y (or C ) S10.001

Intracellular homologous proteases and GPI-anchored proteases in S. cerevisiae are given in the last column. ( Intracellular homologous proteases can also be found in species which secrete proteases). When possible, the proteases are given with their identifier number in the MEROPS data base. MEROPS names of secreted proteases from pathogenic fungi are listed in Table 2. ß: Most studies about proteases secreted by Aspergillus of the flavus group concerned Aspergillus oryzae and A. sojae used in food industry. During fermentation, these moulds secrete a large variety of carbohydrases and proteases, that are essential for efficient solubilization and hydrolysis of the raw materials, soybean or wheat. Molecular typing methods do not allow the differentiation of A. flavus and A. oryzae ( Kumeda and Asao, 1996). In fact, A. oryzae strains used in food fermentation differ from those of A. flavus by not producing aflatoxins but both species were reported as responsible for infections ( Rhodes et al., 1990; Ziskind et al., 1958). ß1: Putative A. fumigatus gene coding for a NpII cloned by Ramesh et al. (1995). ß2: Jousson et al. (unpublished results)

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Protease (sub)family

406

Table 1. Secreted endo- and exoproteases from different human pathogenic fungi.

Secreted proteases from pathogenic fungi Table 2. Identifier and MEROPS names of secreted proteases from pathogenic fungi. Identifier

MEROPS Name

Endo/Exo proteases

A01.012 A01.014 A01.016 A01.025 A01.026 A01.037 A01.038 A04.002

Rhizopuspepsin Candidapepsin Aspergillopepsin I Peptidase E Peptidase F Canditropsin Candiparapsin Aspergillopepsin II

Endo Endo Endo Endo Endo Endo Endo Endo

M28.006 M35.002 M36.001

MEROPS-AA063 peptidase Deuterolysin Fungalysin

Exo Endo Endo

S08.052 S08.053 S09.008 S09.012 S10.006 S10.014

Cerevisin (subtilisin) Oryzin (subtilisin) Dipeptidyl-peptidase IV ( Aspergillus ) Dipeptidyl-peptidase V MEROPS-AA083 peptidase MEROPS-AA085 peptidase

Endo Endo Exo Exo Exo Exo

amino acids. The classification and the nomenclature of all proteases can be found together with information about them in the MEROPS database accessible at http://www.merops.ac.uk/merops/ merops.htm. The proteases are initially classified following their mode of action and their active sites. Aspartic, cystein, metallo, serine, threonine proteases and proteases with unknown catalytic mechanism are recognized. Each protease is then assigned to a family that is a set of homologous enzymes (Tables 1 and 2). These families are identified by a capital letter representing the catalytic type of the peptidases they contain, together with a unique number. Subfamilies are labelled with a second capital letter (for instance S9B and S9C in the serine proteases, Table 1). Different families that are thought to have a common origin are grouped into a clan. The proteases can be further divided into endoproteases (or endopeptidases) and exoproteases (or exopeptidases). The endoproteases cleave peptide bonds internally within a polypeptide. The exoproteases cleave peptide bonds only at the N- or the Cterminus of polypeptide chains. The endo- and exoproteases cooperate very efficiently in protein digestion, in which it can be said that the main function of the former is to produce a large number of free ends on which the latter may act. Protein digestion into amino acids has been thoroughly investigated in bacteria from the genus Lactobacillus (O'Cuinn et al., 1999).

407

Endoproteases secreted by pathogenic fungi Many fungal species secrete proteases when grown in a medium containing protein as sole nitrogen source (see for instance (Reichard et al. 1990; Togni et al., 1991; Hube et al.,1994; Mignon et al., 1998b; Brouta et al., 2001)). Small molecules such as ammonium ions and amino acids repress the genes coding for these proteases. However, many proteases, like several Candida aspartic proteases, are secreted in other conditions (Borg-von Zepelin et al., 1998). The endoproteases, secreted by pathogenic fungi are aspartic proteases of the pepsin family (A1 family), serine proteases of the subtilisin subfamily (S8A), and metalloproteases of two different families (M35 and M36) (see below). To these proteases has to be added (i) the A. niger aspergillopepsin B (PepB or also proteinase A) (Inoue et al., 1991; Takahashi et al., 1991; Mattern et al.,1992) from the A4 family and (ii) a unique chymotrypsin-like protease from Coccidioides immitis (Cole et al., 1992) for which the family S19 was created. Secreted proteases from the trypsin family (S1) are well documented from Fusarium spp. (see MEROPS > S01.103) and insect pathogenic fungi (see MEROPS > S01.103 and MEROPS > S01.412). However, such proteases have, so far, never been identified from culture supernatants of human pathogenic fungi, and expressed sequence tag databases from Candida spp. and Aspergillus spp. also lack S1 homologs. Like many other secreted endoproteases, those from pathogenic fungi are synthesized as precursors in a preproprotein form. The prepeptide or signal peptide (Von Heijne, 1986) is necessary for entering the secretory pathway by transporting the protein across the membrane of the endoplasmic reticulum (Pfeffer and Rothman, 1987). As in bacteria, the further N-terminal extension called the propeptide (30 to 250 amino acids in length) has been found to be essential and specific for assisting the correct folding and the secretion of its associated protein (for review, (Eder and Fersht, 1995)) (Beggah et al., 2000; Schoen et al., 2002). Upon completion of folding, the propeptide is cleaved and removed to generate the active enzyme through an autoproteolytic reaction or, like in the Candida secreted aspartic proteases (Saps), through an exogenous proteolytic reaction in the Golgi apparatus via the membranebound protease Kex2 (Togni et al., 1996; Newport and Agabian, 1997) which specifically cleaves peptides after the tandem sequences KR (Julius et al., 1984).

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In contrast to the secreted endopeptidases, the exopeptidases secreted by pathogenic fungi have generally no propeptide and are made with a signal peptide only. The carboxypeptidases secreted by A. niger which are made as preproproteins are exceptions to this rule (see below). Aspartic proteases The aspartic proteases secreted by fungi (Table 1) are generally similar to pepsin (A1 family) or belong to the A4 family which contains only fungal secreted enzymes. The peptidases of the A1 family are totally inhibited by pepstatin, whereas the enzymes of the A4 family to which the A. niger PepB belongs, are insensitive to this inhibitor. The molecular mass of the fungal secreted aspartic proteases (Sap) of the A1 family is about 40 kDa and their activity is optimal at a pH around 3 to 4, although some proteases have an optimal activity at around pH 5 and are still active at neutral pH (R¸chel et al., 1986; Togni et al., 1991; de Viragh et al., 1993; Borg-von Zepelin et al., 1998; Reichard et al., 1995; Schoen et al., 2002). They are synthesized as preproproteins with a relatively short propeptide of 27 ± 60 amino acids. Their general structural pattern contains 3 highly conserved regions and 4 cysteine residues. Two of the conserved regions with the motifs DSG or DTG contain the two reactive aspartic residues of the active site of the pepsin-like proteases. The third conserved region is found at the C-terminus of the protein. The 4 cysteine residues form two disulfide bridges conserved in all proteases of the pepsin family. Many pathogenic fungi contain a gene family coding for Saps. Ten, four and three genes encoding Saps have been cloned from C. albicans, C. tropicalis and C. parapsilosis, respectively, to date (Togni et al., 1991; de Viragh et al., 1993; Monod et al., 1994; 1998; Zaugg et al., 2001) (unpublished results). However, the C. albicans SAP9 and SAP10 deduced amino acid sequences show a putative GPI anchor at the C-terminus of the translation products. Large gene families coding for secreted aspartic proteases are also present in Rhizopus species (Ohtsuru et al., 1982; Farley and Sullivan, 1998; Schoen et al., 2002; MEROPS > Rhizopus). A. fumigatus secretes one protease called Pep1 (Reichard et al., 1994; 1995) highly similar to the Aspergillopepsin A (PepA) from A. niger (Berka et al., 1990) and PepO from the koji mould A. oryzae (Berka et al., 1993) (Table 1). However, a second aspartic protease was found to be present in the cell wall of A. fumigatus and was called A.

fumigatus Pep2 (Reichard et al., 2000a). The amino acid sequences of Pep2 and the previously characterized secreted A. fumigatus Pep1 are only 27% identical. In fact, Pep2 is more related to the vacuolar proteases of S. cerevisiae (PrA) and Neurospora crassa encoded by the gene PEP4 in both species (Ammerer et al., 1986; Woolford et al., 1986; Vasquez-Laslop et al., 1996), and the intracellular protease of A. niger encoded by the gene pepE (Jarai et al., 1994). To our knowledge, the localization of PepE in A. niger is not yet known. It is tempting to assume that A. fumigatus Pep2 and A. niger PepE are also vacuolar and have the same function in both Aspergillus species. Although it could be argued that the observed binding of Pep2 to the cell wall might be an artefactual cytoplasmic contamination during isolation of the enzyme, there is evidence that Pep2 might be located somewhere other than in the vacuole alone. Indeed, overexpression of the gene PEP4 in S. cerevisiae, and high production of recombinant A. fumigatus Pep2 in P. pastoris led to the presence of enzymes in significant amounts in the culture supernatants (Reichard et al., 2000a). Thus, A. fumigatus Pep2 might be directed not just to the vacuole but also to the fungal cell wall to fulfil additional functions. Cell wall-associated proteases might facilitate hyphal penetration into the proteinrich connective tissue layers of the host. The absence of the enzyme in the A. fumigatus culture supernatant could be either explained by a regularly limited secretion or, alternatively, by a high fungal cell wall capacity for retaining the enzyme. Metalloproteases Metallo-endoproteases secreted by pathogenic fungi belong to two different families, the deuterolysins (M35) and the fungalysins (M36). The enzyme type of the M35 family is the A. oryzae protease described under the name of neutral protease II (NpII) (Nakadai et al., 1973b). This enzyme has a polypeptidic chain of 20 kDa (Tatsumi et al., 1991) and is active at pH 6 to 9 (Rhodes et al., 1990). It is synthesized as a preproprotein with a long propeptide of 280 amino acids (Tatsumi et al., 1991). An orthologous gene encoding a putative deuterolysin was also found in the A. fumigatus genome (Ramesh et al., 1995). Members of the M35 family are found in many fungi but also in the bacterium Aeromonas hydrophyla (for references, see MEROPS > M35 family). The example type of the M36 family is the secreted metalloprotease (Mep) isolated from the pathogenic A. fumigatus (Monod et al., 1993a; 1994; Markaryan et al.,1994; Sirakova et al.,1994). This enzyme

Secreted proteases from pathogenic fungi

409

Table 3. Distribution of proteases belonging to the same families as the secreted proteases from pathogenic fungi. Family A1 A4 M28 M35 M36 S8 S9 S10 S19

Bacteria

Archea

Protozoa

Fungi

Plants

Animals

‡

‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡

‡

‡

‡

‡

‡ ‡ ‡

‡ ‡ ‡

‡ ‡

‡

‡

‡ ‡

‡ ‡

‡ ‡ ‡

Virus

‡

Table 4. Secreted proteases isolated and characterized from dermatophytes. Dermatophyte

Mol. mass (kDa)

References

T. rubrum T. rubrum T. rubrum T. rubrum T. mentagrophytes T. mentagrophytes T. mentagrophytes Different strains of T. mentagrophytes M. canis M. canis M. canis M. canis

34.7 36 44 27 48 38 ± 41 33 28 ± 65 ( One to three enzymes per strain) 45 43.5 33 31.5

Sanyal et al., 1985 Asahi et al., 1989 Asahi et al., 1989 Apodaca et al., 1989 Yu et al., 1968 Tsuboi et al., 1989 Aubaid and Muhsin, 1998 Siesenop and Bˆhm, 1995 Takiuchi et al., 1982, 1984 Brouta et al., 2001 Lee et al., 1987 Mignon et al, 1998b

is highly similar to the A. oryzae neutral protease I (NpI) previously isolated and characterised in the seventies (Nakadai et al., 1973a), and to the 43.5kDa metalloprotease recently isolated from Microsporum canis culture supernatants (Brouta et al., 2001, 2002). These enzymes belong to the same clan as the anthrax lethal factor (MEROPS > M34) and the Bacillus thermoproteolyticus thermolysin (MEROPS > M4). The enzymes of the M36 family have a polypeptidic chain of about 380 amino acids (42 kDa) and are synthesized as preproproteins with long propeptides of about 230 amino acids. A. oryzae NpI and the M. canis metalloprotease are glycosylated whereas A. fumigatus Mep is not (Doumas et al., 1999). These three proteases are active at pH 6 to 9. The fungalysins (M36) are only found in fungi (Table 3). They are unique in Aspergillus species, but are multiple in dermatophytes. Indeed, a family of genes coding for these proteases was recently and for the first time described in M. canis (Brouta et al., 2002) and found in other dermatophyte species such as Trichophyton rubrum and T. mentagrophytes (Capoccia, unpublished results). The two families of metalloproteases M35 and M36 do not share similarity with the exception of

the HEXXH motif characteristic of Zn‡‡ metalloproteases. The proteases of both families are inhibited by agents chelating divalent metallic ions such as EDTA and orthophenanthroline, as well as by phosphoramidon. Serine proteases The serine endoproteases secreted by pathogenic fungi belong to the subtilisin subfamily (S8A) and are totally inhibited by PMSF, antipain and chymostatin but not by inhibitors of other serine proteases such as TLCK and TPCK (Monod et al., 1991; Mignon et al., 1998b). Their molecular mass is about 28 ± 30 kDa and their activity is optimal at around pH 8 to 9. For this reason, they are often called alkaline proteases with the acronym Alp. They are synthesized as preproproteins with relatively long propeptides of 70 ± 100 amino acids. The fungal subtilisins share a high degree of sequence similarity near the His and Ser residues of their active site (Jaton-Ogay et al., 1992). In the nineties, a secreted subtilisin (named Alp for alkaline protease) from the pathogenic A. fumigatus, highly similar to that secreted by A. oryzae (Tatsumi et al., 1989), was described by many authors

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M. Monod et al.

(Reichard et al., 1990; Monod et al., 1991; Frosco et al., 1992; Larcher et al., 1992; Kolattukudy et al., 1993). However, a second subtilisin was also shown to be present in the cell wall of A. fumigatus and was called Alp2 (Reichard et al., 2000b). Alp2 shares 49% identity with the vacuolar protease B of S. cerevisiae and 72% with the A. niger homologue PepC (Frederick et al., 1993) which is not secreted. Again, A. fumigatus Alp2 might be directed not just to the vacuole but also to the fungal cell wall to fulfil additional functions. In particular, the ALP2 gene is required for regular sporulation. Targeted disruption of the ALP2-encoding gene did not significantly affect vegetative growth but led to a reduction of sporulation higher than 80% in the alp2-negative mutants, correlated with a reduction of approximately 50% of the median size of conidiophore vesicles. As with Pep2, the absence of the enzyme from the A. fumigatus culture supernatant could be either explained by a regularly limited secretion or, alternatively, by a high fungal cell wall capacity for retaining the enzyme. Most of the keratinases isolated from dermatophyte culture supernatants (Table 4) are described as serine proteases or, at least, the effects of various inhibitors support the view that they belong to this class of proteases. Gene families in M. canis and T. rubrum coding for serine proteases of the subtilisin family were also recently isolated (Descamps et al., 2002) (Capoccia, unpublished results). The deduced protein sequences showed 50 ± 70% similarity to the homologous protease-encoding genes previously characterized in A. fumigatus. One M. canis gene encoded the 31.5-kDa subtilisin previously isolated and characterized from culture supernatant (Mignon et al., 1998b). GPI-anchored proteases encoded by a gene family with at least 12 members are found at the cell surface of Pneumocystis carinii sphaerocysts (Russian et al., 1999). They are highly similar to the Golgi Kex2 protease and also belong to the subfamily S8B (kexins) of the serine proteases.

Exoproteases secreted by pathogenic fungi Several aminopeptidases, carboxypeptidases and dipeptidyl-peptidases have been isolated from different Aspergillus culture supernatants (Nakadai et al., 1972a ± c; 1973a ± g; Beauvais et al., 1997a, b; Doumas et al., 1998). To date, DNA sequences are available for two A. oryzae genes coding for cobalt-activated aminopep-

tidases belonging to the M28 family (United State patents # 6,127,161 and # 5,994,113). The first enzyme has a polypeptidic chain of 52 kDa, is glycosylated and belongs to the same subfamily M28B as the vacuolar protease Y of S. cerevisiae (MEROPS >M28.001). The second (MEROPS >M28.006) has a polypeptidic chain of 35 kDa, is not glycosylated and belongs to the same subfamily M28A as Aeromonas and Vibrio leucyl aminopeptidases (MEROPS > M28.002). Two genes and a single gene coding for carboxypeptidases of the same family of serine proteases (S10) were cloned from A. niger and A. oryzae, respectively. Both A. niger proteases [carboxypeptidase I (MEROPS > S10.014) and carboxypeptidase II or carboxypeptidase F (MEROPS > S10.006)] are 471 and 481 amino acids long proteins (Svendsen and Dal Degan, 1998) and are made as preproproteins (van den Homberg et al., 1994). In contrast, the A. oryzae carboxypeptidase (550 amino acids long) has no (cleaved) prosequence (Blinkovsky et al., 1999). The A. oryzae and the A. niger carboxypeptidases II are closely related to the vacuolar carboxypeptidase Y (or carboxypeptidase C) of S. cerevisiae (MEROPS > S10.001). Two different secreted dipeptidyl-peptidases called DppIV and DppV have been characterized at the biochemical and molecular level in A. fumigatus and A. oryzae (Beauvais et al., 1997a, b; Doumas et al., 1998) (Doumas et al., unpublished results). They are serine proteases belonging to two different subfamilies (MEROPS S9B and S9C). These enzymes have an apparent molecular mass of about 90 kDa, and contain approximately 10 kDa of N-linked carbohydrates. Like the homologous human membrane-bound DppIV (or CD26), the DppIV of both Aspergillus species (subfamily S9B) releases X-Pro and to a lesser extent X-Ala dipeptides from the N-terminal extremity of peptides (Beauvais et al., 1997b; Doumas et al., 1998). The DppV (subfamily S9C) releases X-Ala, but also His-Ser and Ser-Tyr, dipeptides from the N-terminal extremity of peptides (Beauvais et al., 1997a). The DppV from different species of Aspergillus was first identified (Biguet et al., 1967) as one of the two major antigens used in the serodiagnosis of aspergillosis, and was wrongly called chymotrypsic antigen because of its ability to degrade, like chymotrypsin, the chromogenic substrate N-acetylphenylalanine naphthyl ester (NAPNE). Due to its biochemical properties, a new class of dipeptidylpeptidases (DppV) was created (Beauvais et al., 1997a). Homologous DppVs were described in Trichophyton tonsurans and T. rubrum (Woodfolk et al., 1996; 1998). Recombinant T. rubrum DppV

Secreted proteases from pathogenic fungi

could be produced in large amounts using P. pastoris. T. tonsurans DppV elicited immediate hypersensitivity and delayed-type hypersensitivity skin test reactions in humans.

Role of proteases in virulence During recent years, secreted proteolytic activities have attracted a lot of attention as potential virulence factors in fungi. However, most of the investigations concerned C. albicans and A. fumigatus. The Sap family of C. albicans is one of the virulence traits which make this species more adapted than others to overcome the residual host barriers for causing mucosal infections and deep mycoses. It is also generally accepted that secreted keratinases are important for the virulence of dermatophytes. For A. fumigatus the role of proteolytic activity is more questionable. The importance of the secreted proteases for C. albicans and related species, A. fumigatus and the dermatophytes is discussed below. Cryptococcus neoformans (Brueske, 1986; Aoki et al., 1994; Chen et al., 1996; Ruma-Haynes et al., 2000), Paracoccidioides brasiliensis (Carmona et al., 1995; Puccia et al., 1998; de Assis et al., 1999) and Histoplasma capsulatum (Okeke and Muller, 1991; Muotoe-Okafor et al.,1996) were shown to secrete proteolytic activity in vitro. However, an individual protease has not yet been isolated and characterized. The role of secreted proteases in virulence of these fungi therefore remains putative.

Candida spp. The occurrence of Candida Saps has been demonstrated on the surface of fungal elements colonising mucosa and penetrating tissues (Borg and R¸chel, 1988). The fact that C. albicans possesses at least 10 SAP genes and inhabits diverse host niches leads to the question whether different Saps are expressed by C. albicans in reaction to specific environmental conditions. This question has been addressed by a number of laboratories using a variety of approaches including immunofluorescence microscopy using specific antibodies (R¸chel et al., 1991; Borg-von Zepelin et al., 1998), Northern blotting analysis (De Bernardis et al., 1995), RT-PCR (in vitro and in vivo) (Schaller et al., 1998; 1999; Naglik et al., 1999), and a gene expression reporter system (Staib et al., 1999, 2000). Additionally, several groups have also assessed the effects of knock-out gene mutations in

411

both individual and multiple SAP genes (Schaller et al., 1999; Hube et al., 1997; Sanglard et al., 1997; De Bernardis et al., 1999; Kretschmar et al., 1999). Altogether, many experimental studies have shown the importance of the three closely related Sap1, Sap2 and Sap3 in adherence, and the importance of the three closely related Sap4, Sap5 and Sap6 in the establishment of deep-seated candidiasis (for a review, see (Hube and Naglik, 2001)). In particular, the three latter closely related isoenzymes, which are optimally active at pH 5, are secreted by C. albicans after phagocytosis by peritoneal macrophages. They could act as cytolysins being involved in the direct destruction of intracellular components of the macrophages. Interestingly, Sap1, Sap2 and Sap3 which are apparently involved in adherence were inhibited by HIV protease inhibitors (Borg-von Zepelin et al., 1999). An in vitro assay was developed to test the effect of different drugs on adherence of different Candida species to epithelial cells (Borg-von Zepelin et al., 1997; 1999). Immunofluorescence microscopy allowed the demonstration of Sap secretion by C. albicans, C. dubliniensis and C. tropicalis after interaction with the target cells during the adherence process (Borg-von Zepelin et al., 1997, 1999) (unpublished results). The influence of HIV protease inhibitors on the adherence of various C. albicans strains to epithelial cells was correlated to the direct effect of the HIV protease inhibitors on the different Saps (Borg-von Zepelin et al., 1999; Korting et al., 1999) leading to the hypothesis that the decrease in the frequency of oropharyngeal candidiasis might be assisted by inhibition of Saps involved in C. albicans adherence (for a review, see (Munro and Hube, 2002)). In another study, Indinavir¾ and Ritonavir¾ were shown to have a good anti-C. albicans effect in a rat vaginitis model (Cassone et al., 1999). Therefore, the development of specific aspartic protease inhibitors might be of interest for the treatment of mucosal candidiasis. Moreover, it is possible to block adherence and invasion events of C. albicans in mice with sublethal doses of pepstatin A, a specific inhibitor of aspartic proteases (Ollert et al., 1993; Ray and Payne, 1988; R¸chel et al., 1990; Fallon et al., 1997). As in C. albicans, C. dubliniensis Saps seem to be important for the establishment of mucosal infection. C. dubliniensis has been recovered predominantly from the oral cavities of human immunodeficiency virus (HIV)-infected individuals and AIDS patients often receiving fluconazole prophylaxis or therapeutic treatment for oropharyngeal candidiasis (Coleman et al., 1997; Sullivan et al., 1997). In the presence of fluconazole, C. dubliniensis showed an

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increase in adherence to epithelial cells while protease antigen was more strongly expressed (Borg-von Zepelin et al., 2002). Increased adherence of C. dubliniensis strains in the presence of fluconazole could explain its high rate of recovery in HIV-patients in the last few years. Secreted proteolytic activity is also important for adherence of C. tropicalis most commonly not involved in thrush, but causing disseminated candidiasis in patients with leukaemia and prolonged neutropenia (Kontoyiannis et al., 2001). A particular strain deficient in Sap secretion (C. tropicalis DSM 4959) was less adherent than the other protease-secreting C. tropicalis strains when compared in parallel tests using the developed in vitro adherence assay. However, C. tropicalis DSM 4959 could be rendered more adherent by adding Candida Sap extracts from different sources (Borg-von Zepelin, unpublished results).

Aspergillus fumigatus To determine the importance of the secreted A. fumigatus endoproteases in virulence, different mutants constructed by targeted gene disruption were tested for pathogenicity in murine or guinea pig models (Monod et al., 1993b; Tang et al., 1993a, b; Jaton-Ogay et al., 1994; Reichard et al., 1997). In all experiments with single alp1, mep, pep1 mutants and double alp1, mep mutants (the latter totally deficient in proteolytic activity at neutral pH in vitro) no difference in pathogenicity was observed between the wild-type strains and the mutants. Mortality curves were not statistically different and histopathological studies of lungs from infected mice showed a similar extent of mycelial growth in parental and mutant strains. Invasion of the lung tissues by fungus occurred with the wild-type and mutant strains through the bronchial epithelium after extensive development of the fungus inside the bronchia and bronchioles. When the collagen and elastin matrix was crossed by fungal hyphae no apparent proteolytic degradation was seen around the fungus (Jaton-Ogay et al., 1994). Therefore, it must be concluded that the secreted A. fumigatus proteolytic activity identified in vitro at neutral and acidic pH, due to ALP1, MEP and PEP1, is not essential for the invasion of tissue. One could object to this somewhat surprising conclusion a discrepancy between animal models and human infection. It is also possible to object that other proteases are produced by induction through specific host factors. However, other arguments are in favour for the absence of a role of the A. fumigatus secreted proteases in pathogenicity: (i) the lack of

vessel wall elastolysis in human invasive aspergillosis demonstrated with tissues obtained at autopsy (Denning et al., 1992), and (ii) the fungal virulence not being attenuated by monoclonal antibodies able to inhibit ALP (Frosco et al., 1994). Consequently, it is reasonable to concede that secreted proteolytic activity is not sufficient to explain the pathologic behaviour of A. fumigatus and it is necessary to search elsewhere for other factors to elucidate the development of the fungus in the human host. A. fumigatus is the archetypal opportunistic fungus, which is saprophytic in the environment and does not invade immunocompetent people. This fungus grows rapidly at 37 8C on rich and minimal medium. One could consider that A. fumigatus is capable of finding a particular ecological niche in the bronchia. This niche would be characterized by nutrient and temperature conditions favourable to its saprophytic growth since this fungus accounts for only a small proportion of the airborn mold spores, even though it is the most frequently isolated fungus from lung and sputum (Mullins and Seaton, 1978; Schmitt et al., 1991). Infection of lung tissues by A. fumigatus occurs when the host phagocytic defences have been weakened by immunosuppressive treatments.

Dermatophytes A biological characteristic of the dermatophytes is their ability to invade keratinized tissues, and all investigated dermatophytes produce proteolytic activity in vitro. There are many works reporting the isolation and characterization of one or two secreted proteases from an individual species of dermatophyte (Table 1). The proteases secreted from dermatophytes are generally described as keratinases without paying attention to the cornified cell envelope made of other components. Indeed, the keratinized tissues such as the epidermis, nails, and hair are not only made of keratins but also by an insoluble network of different crosslinked proteins such as involucrin, loricrin, and small proline-rich proteins which form the cornified cell envelope (Simon and Green, 1985; Hohl et al., 1995; Steinert and Marekow, 1995; 1997). To date, there has been no study comparing proteases isolated from different dermatophytes, which could explain the relative specificity of these fungi in causing different types of dermatophytosis. Furthermore, with the exception of a M. canis 31.5-kDa serine protease (Mignon et al., 1998b) and a 43.5kDa metalloprotease (Brouta et al., 2001), there are no reported amino acid sequences of the N-terminus

Secreted proteases from pathogenic fungi

of these proteins to further identify and characterize the isolated extracellular enzymes. Recent investigations have shown that proteases secreted by dermatophytes are similar to those of other fungi such as Aspergillus spp. but with a different substrate specificity and organized in gene families (Descamps et al., 2002; Brouta et al., 2002) (Capoccia, unpublished results). Both M. canis 31.5kDa serine protease and 43.5-kDa metalloprotease are able to digest keratin azure. The intron-exon structures of the isolated genes were shown to be similar to the homologous genes isolated from A. fumigatus and A. oryzae. These results are not surprising since the teleomorphs of Aspergillus species and the teleomorphs of dermatophyte species are closely related. Indeed, they belong to the same taxonomic group of Ascomycetes producing prototunicate asci in cleistothecia (class Eurotiomycetes). Large protein families are known to be specific virulence traits of microorganisms. It is the case for the cell surface agglutinin-like proteins (Hoyer et al., 1995; Hoyer and Hecht, 2000) and the secreted aspartic proteases of C. albicans. In particular, the ability of dermatophytes to degrade proteins of the skin may require the use of combinations of different specific proteases. In vivo expression of M. canis 31.5-kDa serine proteinase and 43.5-kDa metalloprotease (Mignon et al., 1998b; Brouta et al. 2001) was demonstrated in both naturally infected cats and experimentally infected guinea pigs.

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Differential substrate specificity of fungal secreted proteases It is evident that the dermatophyte secreted proteases use proteins of the cornified cell envelope as a substrate since these fungi grow exclusively in the stratum corneum, nails or hair as sole nitrogen and carbon sources. Characterization of these proteases could explain the relative specificity of the species in causing different types of dermatophytosis. Human stratum corneum, fibronectin, laminin and mucin were also shown to be degraded by C. albicans secreted proteolytic activity (Negi et al., 1984; Colina et al., 1996; Morschh‰user et al., 1997). In addition to a function of direct degradation of structural proteins of the host, the fungal secreted proteases can have deleterious effects on the immune system, activate other host proteases and inactivate host peptidic protease inhibitors and biological peptides (Table 5). The Candida Saps can interact with the immune system by acting as possible cytolysins in macrophages (Borg-von Zepelin et al., 1998), by cleaving immunoglobulins (R¸chel et al.; 1982, R¸chel, 1986; Kaminishi et al., 1995) and complement C3 protein (Kaminishi et al., 1995), and by activating proinflammatory cytokines (Beausejour et al., 1998). Activation of bovine blood coagulation factors was performed at slightly acidic pH in vitro

Table 5. Humoral and host defence proteins and biological peptides cleaved by secreted proteases from pathogenic fungi. Function

Activation of host proteases

Inactivation of protease inhibitors

Effects on cytokines and biologic peptides

Substrate

Protease

Reference

Albumin Immunoglobulins

CaSaps CaSap2

R¸chel et al., 1982 R¸chel et al., 1982 R¸chel, 1986 Kaminishi et al., 1995

Complement C3

CaSap2

Kaminishi et al., 1995

Bovine factor X

CaSap2

Human factor XII Bovine prothrombine a2 Macroglobulin

Rhizopuspepsin CaSap2 CaSap2 CaSap2

R¸chel, 1983 Kaminishi et al., 1994 Schoen et al., 2002 Kaminishi et al., 1994 Kaminishi et al., 1994

a1 Proteinase inhibitor Cystatin A Interleukin 1b Endothelin-1 precursor Neuropeptide Y Glucagon-like peptide-1-(7-36) amide [des-Arg1] Bradykinin

CaSap2 CaSap2 CaSap2 CaSap2 Aspergillus DppIV Aspergillus DppIV Aspergillus DppIV

R¸chel et al., 1982 Kaminishi et al., 1995 Kaminishi et al., 1995 Tsushima et al., 1994 Beausejour et al., 1998 Tsushima and Mine, 1995 Beauvais et al., 1997b Beauvais et al., 1997b Beauvais et al., 1997b

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with C. albicans Sap2 (R¸chel, 1983; Kaminishi et al., 1994) and with a Rhizopus secreted aspartic protease (Schoen et al., 2002). It was recently shown that human plasma fibronectin contains cryptic and latent aspartic, metallo and serine proteases (Unger and Tschesche, 1999; Schnepel and Tschesche, 2000). These proteases can be generated and activated in the presence of Ca‡‡ from the fragments produced by cathepsin D digestion of fibronectin. It is conceivable that fungal secreted proteases may activate such cryptic proteases. By inactivating peptidic protease inhibitors such as a2 macroglobulin, a1 proteinase inhibitor and cystatin, the fungal secreted proteases may also facilitate debilitation of the infected host. The precise function of Candida Saps in the adherence process is not known but two hypotheses can be advanced: (i) the Candida Saps could act as ligands to surface proteins of the specific host cells, which does not necessarily require their enzymatic activity. (ii) The Candida cells use Saps as active enzymes to affect target structures of their host cells leading to conformational changes of surface proteins or ligands of epithelial cells allowing a better adherence of the yeasts. Although it is now clear that proteases may act differently as virulence factors, knowledge on protease substrate specificities remains rather poor and few studies have focused on the research of specific inhibitors. The observed differences of residue specificities at the P1 and P1' sites among Saps (Koelsch et al., 2000) may have some physiological importance since these enzymes have different human protein targets in vivo. Knowledge of substrate specificities will permit us to learn more about the specific action of each protease secreted by pathogenic fungi and to determine their specific contribution to virulence. As mentioned above, the development of specific aspartic protease inhibitors might be of interest for the treatment of mucosal candidiasis. The proteases secreted by dermatophytes could also serve as basis for investigations into new drug targets. Acknowledgements. We thank Dr. H. Pooley and Dr. E. Grouzmann for critical review of the manuscript and assistance with the English. This work was supported by the Swiss National Foundation for Scientific Research, grant 3200-63687.

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