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

Abstract

Pathogenic yeasts of the genus Candida produce secreted aspartic proteinases, which are known to enhance virulence. We focused on Sapp1p proteinase secreted by Candida parapsilosis and studied the final stage of its passage through the cell wall and release into the extracellular environment. We found that Sapp1p displays enzyme activity prior to secretion, and therefore, it is probably fully folded within the upper layer of the cell wall. The positioning of cell surface-associated Sapp1p was detected by cell wall protein labeling using biotinylation agents, extraction of cell wall proteins by β-mercaptoethanol, immunochemical detection, and mass spectrometry analysis. All lysine residues present in the structure of soluble, purified Sapp1p were labeled with biotin. In contrast, the accessibility of individual lysines in cell wall-associated Sapp1p varied with the exception of four lysine residues that were biotinylated in all experiments performed, suggesting that Sapp1p has a preferred orientation in the cell wall. As the molecular weight of this partially labeled Sapp1p did not differ among the experiments, we can assume that the retaining of Sapp1p in the cell wall is not a totally random process and that pathogenic yeasts might use this cell-associated proteinase activity to enhance degradation of appropriate substrates.

Figure 1

Western blot detection of Sapp1p associated with the cell surface of C. parapsilosis. Panel A: fractions obtained by cell surface washing with PTB buffer; Panel B: fractions from washing with PTB containing 0.5% βME. Lane 1, fraction after 10-min incubation; Lanes 2–5, fractions after repeated washings. Detection was performed using polyclonal antibodies raised against Sapp1p.

Figure 2

Profile of proteins isolated from the C. parapsilosis cell wall with 1% βME. Lane 1, silver-stained SDS-PAGE; Lane 2, Western blot of cell wall protein samples detected with polyclonal antibodies raised against a peptide corresponding to part of the Sapp1p sequence (anti-Sapp1p/186-199).

Figure 3

Cleavage of the fluorescent substrate Dabcyl-EHVKLVE-EDANS by cell-associated Sapp1p. Panel A: cleavage products obtained from incubation of C. parapsilosis cells with the substrate. Panel B: substrate cleavage products from incubation of C. parapsilosis cells in the presence of 1 μM pepstatin A. Arrows indicate the position of Sapp1p-specific cleavage product. The position of the cleavage product was verified by in vitro substrate cleavage with purified Sapp1p.

Figure 4

Western blot analysis of stepwise biotinylation of purified Sapp1p using a 4-, 8-, 12-, 16-, or 32-fold molar excess of biotinylation reagent. The blot was probed with anti-Sapp1p polyclonal antibodies (anti-Sapp1p/186-199). Panel A: biotinylation performed with sulfo-NHS-biotin; Panel B: biotinylation performed with sulfo-NHS-LC-biotin. Sapp1p, purified nonbiotinylated proteinase; b-Sapp1p, purified fully biotinylated proteinase.

Figure 5

Western blot analysis of biotinylation of intact C. parapsilosis cells with sulfo-NHS-biotin (left) or sulfo-NHS-LC-biotin (right) using polyclonal anti-Sapp1p antibodies. Lane 1, unbiotinylated Sapp1p; Lanes 2, 5, βME extract after biotinylation of the intact cells; Lanes 3, 6, material released from the cell surface during incubation with biotinylation reagents; Lanes 4, 7, purified, fully biotinylated Sapp1p.

Figure 6

Positions of lysine residues in the three-dimensional (Panel A) and primary (B) structures of Sapp1p. The unbiotinylated lysine residues are indicated in red, the biotinylated lysines in green, and the undetected lysine residue (Lys24) in black. The sequence regions obtained from the combined in-gel digest of biotinylated Sapp1p are framed in gray in panel B.