Candida parapsilosis is an opportunistic fungal pathogen responsible for approximately 30% of candidaemia episodes in new-borns, and 10%–15% of Candida infections in adults worldwide. Fungal extracellular hydrolytic enzymes, especially secreted aspartyl proteinases (SAP) are important virulence factors of Candida species, that contribute to the development of disseminated candidiasis. SAPs cause significant damage to epithelial cells, promote the escape of fungal cells during host attacks by cleaving host complement proteins and also modulate the secretion of inflammatory cytokines. Although several studies have investigated the role of C. albicans secreted aspartyl proteinases in its virulence, less is known about the precise role of C. parapsilosis secreted aspartyl proteinases (SAPPs) in host invasion, especially during complement evasion. Thus, we aimed to study the function of C. parapsilosis secreted aspartyl proteinases during host attack.
To investigate the role of SAPP genes, a deletion mutant strain (sapp-/-) was generated, lacking all functional CpSAPP ORFs. To examine the effect of complement proteins on the growth of the C. parapsilosis GA1 (wild-type) and sapp-/- strains, their growth was measured in YPD and YCB liquid medium supplemented with 20% of either normal human plasma (NHP) or heat-inactivated human plasma (HiP) at 30 and 370C. In order to examine the effect of gene deletion on viability and appearance, we also tested their growth on different complex, minimal and stressor containing media.Virulence properties of the mentioned strains were also tested using an epithelial cell line, the human monocytic cell line THP-1 and human macrophages (PBMC-DMs). The performed assays and methods included the LDH assay, MTT assay, RT-PCR, FACS and ELISA. Protease activity of the wild-type and sapp-/- strains was also examined by SDS-PAGE. In silico analyses were also performed to evaluate efficiencies of HIV protease inhibitors against Sapp’s.
Growth rates of the sapp-/- strain were similar to that of the wild-type strain in both YPD and YCB medium regardless of the temperature used, whereas addition of NHP significantly inhibited the growth of the mutant strain only. Deletion of CpSAPPs did not affect biofilm formation, nor the morphological attributes of C. parapsilosis. Although, the sapp-/- strain showed significant differences in its virulence when compared to the wild-type. THP-1 and PBMCDM’s killed and phagocytosed sapp-/- cells more efficiently. Mutant cells also induced less damage to human epithelial cells, THP-1 cells and to PBMC-DMs compared to the wild-type strain. Furthermore, when examining host cytokine responses, the wild-type strain induced higher levels of IL-1β, TNF-α and IL-8 than the sapp-/- mutant strain. In addition, extracellular proteinases derived from the wild-type strain’s supernatants efficiently cleaved bovine serum albumin whereas no such activity was observed in case of the sapp-/- mutant strain. Our in silico data suggested that several HIV proteases could be potential inhibitors of C. parapsilosis extracellular proteinases.
Our preliminary data suggest that CpSAPPs do not affect fungal viability, morphology or biofilm formation, however significantly contribute to the virulence of C. parapsilosis in vitro, thus play an important role in C. parapsilosis virulence.

This research project was supported by NKFIH K123952.

The role of Candida parapsilosis secreted aspartic proteases in the regulation of inflammation and immune evasion