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 b-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.

Extracellular proteinases of Candida species pathogenic yeasts

“…however, Sapp1 has also been suggested to be attached to the cell wall. 40 Putative sequence motives for Kex2 processing have also been identified in the proforms of C. tropicalis Sapts. 31 These proteases are predominantly extracellular.…”

“…Mature Sapp1 and Sapp2 do not possess potential glycosylation sites . Both Sapp1 and Sapp2 are secreted as soluble enzymes; however, Sapp1 has also been suggested to be attached to the cell wall . Putative sequence motives for Kex2 processing have also been identified in the proforms of C. tropicalis Sapts .…”

Candida parapsilosis: from Genes to the Bedside

“…These cell wall moonlighting proteins have also been identified as being immunogenic in C. albicans, with the exception of Idh2 (219). Other CWPs could have a role in nutrient acquisition or interaction with host components, such as the cell wall adsorbed aspartic proteinase 1 (Aspp1) or an ectophosphatase, as they influence adhesion to epithelial cells (220,221).…”

More than Just Protein Degradation: The Regulatory Roles and Moonlighting Functions of Extracellular Proteases Produced by Fungi Pathogenic for Humans

“…For C. parapsilosis , with the use of mutant strains with the expression of only a single aspartic proteinase—Sapp1, Sapp2, or Sapp3, the participation of Sapp1 and Sapp2, but not Sapp3, in the binding of C. parapsilosis to the TR146 oral epithelial cell line was demonstrated; however, the mechanisms of these interactions require further elucidation [ 31 ]. Of these secreted enzymes, Sapp1 was indicated as being temporarily embedded within the C. parapsilosis cell wall, and capable of operating in the immediate vicinity of the fungal cells; therefore, its functionality may be similar to those described for Sap9 and Sap10 of C. albicans ; nonetheless, this issue requires further research [ 32 ]. For C. glabrata , a family of eleven GPI-linked aspartyl proteases, referred to as yapsins (CgYps) were identified, and their similarity to C. albicans Sap9 and Sap10 proteases was also proposed ( Figure 2 ) [ 33 , 34 , 35 ].…”