Secreted aspartic proteases of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida lusitaniae

Abstract

The frequency of Candida infections has increased in recent years and it has been accompanied by a significant rise in morbidity and mortality. The secretion of aspartic proteases by Candida spp. was demonstrated to be one of the virulence determinants. Candida albicans is classified as the major human pathogen in the genus Candida. However, other species of this genus have been found to cause an increasing number of candidiases. We isolated secreted aspartic proteases (Saps) of C. albicans (Sap2p), C. tropicalis (Sapt1p), C. parapsilosis (Sapp1p), and C. lusitaniae (Saplp) from culture media. All the isolated proteases were N‐terminally sequenced. Their specific proteolytic activities and sensitivity to series of peptidomimetic inhibitors modified in the type of scissile bond replacement as well as in the N‐ and C‐termini were analyzed. The most divergent substrate specificity was observed for the Sap of C. tropicalis. The specificity of Sap of C. lusitaniae is most closely related to that of Sap of C. parapsilosis. We designed and prepared an inhibitor containing phenylstatine isoster that was equipotent towards all four proteases within the range of 10−10−10−9 m. The HIV‐1 protease inhibitors ritonavir, saquinavir, indinavir, and nelfinavir were also tested for the inhibition of four Saps. Only ritonavir and saquinavir inhibited Sap2p, Sapt1p, Sapp1p, and Saplp in micromolar concentrations.

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).…”

Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

“…In this study, we focused on the C. parapsilosis enzyme Sapp1p, which is induced in a similar manner as Sap2 of C. albicans but displays certain enzymological and structural differences. 23,26 To study the passage of Sapp1p through the yeast cell wall during secretion into the extracellular The band containing Sapp1p was cut from a gel and digested with trypsin or chymotrypsin (see Materials and Methods for details). B, number of peptide fragments containing bitonylated residue; S, total number of relevant peptide fragments; n.d., peptide fragment was not detected.…”

“…15 To examine whether cell wall-associated Sapp1p can cleave substrates present in the extracellular space, we incubated thoroughly washed C. parapsilosis cells with a fluorescent peptide substrate for 30 minutes. The substrate was readily hydrolyzed, and its cleavage was sensitive to the presence of pepstatin A, a potent inhibitor of Sapp1p 23 (Fig. 3).…”

The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

“…The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir for these compounds and Saps from several pathogenic Candida species 10,11,12 . While pepstatin A and its analogs inhibit Saps in nanomolar concentrations, inhibition constants for Saps and HIV PR inhibitors ritonavir, saquinavir, indinavir and nelfinavir are within the micromolar range or higher.…”