Secreted Aspartic Proteinase Family of Candida tropicalis

Abstract

Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis EMBL3 genomic library. All bands identified by Southern blotting of EcoRI-digested C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicalis cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other SAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.

Partial Characterization of an Acidic Protease from Rhizopus stolonifer RN-11

“…The acidic protease was purified 41.8-fold and the recovery yielded 24.4% after Sephadex G-75 chromatography ( Table 1). Acidic proteases of varying molecular weights were previously isolated from bacteria, including an aspartate protease from Phycomyces blakesleeanu (35 kDa) [23], aspartate protease from Rhizopus oryzae (34 kDa [11] and 47.5 kDa [24] variants), protease A(37 kDa) and protease B (34 kDa) from Aspergillus niger [25], Aspartic protease Sapt1p (44 kDa), Sapt2p (49 kDa), Sapt3 (55 kDa proproitein and 48 kDa mature protein) [10], and two forms of acid protease (M1 150 kDa and M2 60 kDa) from Aspergillus oryzae [26]. Unlike previously reported data, the purified acidic protease in this study was a monomer with an MW of ~70 kDa according to SDS-PAGE analysis (Fig.…”

“…Several studies have isolated acidic proteases from different fungi, including Aspergillus niger [2,3], Aspergillus oryzae [4][5][6], Penicillium spp. [7,8], Mucor pusillus [7,9], Candida albicans [10], and Rhizopus spp. [11].…”

Infecções oportunistas por leveduras e perfil enzimático dos agentes etiológicos

“…Zaugg e cols 31 avaliaram atividade desta enzima por espécies não-albicans, não sendo observadas variações. Diferenças ou ausência na expressão desta enzima por leveduras isoladas de pacientes HIV-positivos podem ser explicadas pelo uso extensivo de inibidores de proteases 32 .…”

Phylogeny and evolution of the aspartyl protease family from clinically relevant Candida species

“…The mature enzyme is obtained after a successive proteolytic processing performed by a signal peptidase and the Kex2 enzyme (Hube & Naglik 2002). Ten aspartyl proteases have been identified in C. albicans (Sap1-Sap10), four in C. tropicalis (Sapt1-Sapt4), eight in C. dubliniensis (Sapcd1-Sapcd4; Sapcd7-Sapcd10) and three in C. parapsilosis (Sapp1-Sapp3) , Pichova et al 2001, Zaugg et al 2001). Sap9 and Sap10 of C. albicans have C-terminal consensus sequences for glycosylphosphatidylinositol (GPI) modification, similar to yapsin proteins (Monod et al 1998).…”