The in vitro and keratinocyte (HaCAT cells) culture expression of four putative genes coding for secreted aspartyl proteases of Candida dubliniensis-CdSAP1, CdSAP2, CdSAP3, and CdSAP4 (CdSAP1-4) - is reported for the first time. In addition, CdSAP7, 8, 9, and 10, orthologous genes of Candida albicans, were recognized in C. dubliniensis genome. There are no orthologs of C. albicans SAP5 and 6 in C. dubliniensis. The expression of CdSAP1 and 2 was independent of the morphological stage of C. dubliniensis; they are expressed at both pH 4 and pH 7, and were induced with albumin as nitrogen source. CdSAP3 expression was regulated by the pH, and was related to the infection process of keratinocytes. Expression of CdSAP4 predominated during the mycelial phase and the initial stage of keratinocyte infection. During infection of the HaCaT cell line, only genes CdSAP3-4 were expressed, and keratinocytes were affected in their number and shape by the infection with C. dubliniensis; however, this effect decreased in the presence of pepstatin A (aspartyl protease inhibitor). Pepstatin A was not able to inhibit keratinocyte damage. Based on the aforementioned, we suggest that the Saps from C. dubliniensis could be considered a virulence factor just as those from C. albicans, and participants in the nitrogen metabolism of the yeast for nutrient acquisition.

Proteases of Candida dubliniensis have been scarcely studied, these enzymes may play an important role in nitrogen metabolism, post-translational processing, nutritional stress, dimorphism, virulence, etc. In this work, we report the presence of five different intracellular proteases and one extracellular proteolytic activity. The intracellular proteases are: aminopeptidase ycdAPE, carboxypeptidase ycdCP, dipeptidyl aminopeptidase ycdDAP, proteinases ycdPrA and ycdPrB, and extracellular protease Sap activity, measured under several nutritional conditions. C. dubliniensis produced the highest level of intracellular proteolytic enzymes, i.e., ycdAPE, ycdCP, ycdDAP, ycdPrA and ycdPrB in media with peptone during stationary growth phase. Chelating agents affected mainly APE activity; whereas ycdCp, ycdDAP, and ycdPrB were affected by serine protease inhibitors; ycdPrA was affected by pepstatin, an aspartyl protease inhibitor. We found Sap activity in C. dubliniensis in YCB-SBA medium, this activity was inhibited by pepstatin inhibitor. Southern analysis revealed the presence of at least four genes encoding Sap in the C. dublinienisis genome (using as probes SAP1, SAP2, SAP3, and SAP4-6 genes from C. albicans).