Impact of pH, Temperature and Exogenous Proteins on Aspartic Peptidase Secretion in Candida auris and the Candida haemulonii Species Complex

<i>Candida</i> species commonly secrete aspartic peptidases (Saps), which are virulence factors involved in nutrient acquisition, colonization, tissue invasion, immune evasion and host adaptation. However, the regulation of Sap production remains poorly characterized in emerging, widespread and multidrug-resistant members of the <i>Candida haemulonii</i> clade (<i>C. auris</i>, <i>C. haemulonii</i>, <i>C. haemulonii</i> var. <i>vulnera</i> and <i>C. duobushaemulonii</i>). This study investigated the influence of temperature, pH and protein substrate on Sap production using bloodstream isolates of the <i>C. haemulonii</i> clade. Sap activity was initially assessed using the enzyme coefficient (Pz) in fungal cells grown on yeast carbon base (YCB) agar supplemented with bovine serum albumin (BSA) to determine optimal conditions for enzymatic production. <i>C. auris</i> and <i>C. duobushaemulonii</i> exhibited the highest Sap activity at 96 h, pH 4.0-5.0, and 37 °C, whereas <i>C. haemulonii</i> and <i>C. haemulonii</i> var. <i>vulnera</i> displayed more variable and isolate-dependent profiles. Sap production was markedly suppressed at pH 6.0. The addition of pepstatin A, an inhibitor of aspartic peptidases, abolished Sap activity and impaired fungal growth in a dose-dependent manner, confirming both the enzymatic identity and its critical role in nitrogen acquisition. Conversely, YCB supplemented with an inorganic nitrogen source (ammonium sulfate) supported fungal growth but did not induce Sap production. To explore substrate specificity, YCB was supplemented with a panel of proteins. Serum albumins (bovine and human) induced the highest Sap production, followed by globulin, gelatin, hemoglobin, collagen and immunoglobulin G, while elastin and mucin elicited the lowest Sap production. Isolate-specific preferences for protein substrates were observed. Finally, fluorometric assays using a Sap-specific fluorogenic peptide substrate confirmed the presence of Sap activity in cell-free supernatants, which was consistently and entirely blocked by pepstatin A. These findings highlight inter- and intraspecies variability in Sap regulation among <i>C. haemulonii</i> clade, stressing the critical roles of substrate availability, pH and temperature in shaping fungal adaptation to host environments.