Two aspartic proteinases secreted by the pathogenic yeast Candida parapsilosis differ in expression pattern and catalytic properties

Abstract

Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1, SAPP2, and SAPP3. We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1 p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1 p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.

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

“…The fluorescent substrate that we used for the activity assay can differentiate between the Sapp1p and Sapp2p izoenzymes. 15 However, no Sapp2p proteolytic activity was detected in the reaction mixture, which correlates with very low level of Sapp2p in the cell wall detected by Western blot. To confirm that the substrate cleavage was mediated by the cell wall-associated enzyme and not by Sapp1p released into the reaction mixture during the incubation, we preincubated the washed cells in the proteinase activity buffer for 30 min, removed the cells, and incubated the cell-free solution with the proteinase substrate for an additional 30 min.…”

“…This is not surprising, since the concentration of Sapp2p in medium upon induction is always at least one order of magnitude lower than that of Sapp1p. 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.…”

“…For Sapp2p detection, we used antibodies raised against a unique sequence (residues 389-404 of Sapp2p). 15 The protein bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).…”

“…14,15 Analysis of the cleavage products was performed using an HPLC system equipped with an Agilent C-18 column and a fluorescence detector set at an excitation wavelength of 350 nm and an emission wavelength of 480 nm. A linear methanol gradient was used for elution.…”

“…Sapp1p, the main secreted isoenzyme of C. parapsilosis, has a broad substrate specificity and higher catalytic activity than Sapp2p, which has been shown to be secreted constitutively under all conditions tested but in a concentration at least one order of magnitude lower. 15 The SAP genes of pathogenic Candida spp. encode pre-pro-enzymes that consist of a leader peptide followed by a pro-peptide and mature protease.…”