Role of secreted aspartic proteases in Candida albicans pathogenesis

Candida. albicans is the key pathogen for oral candidiasis, and possesses comprehensive virulence mechanisms to cause fungal infection in a host under proper predisposing conditions. The secreted aspartic protease (Sap) family (which consists of 10 family members: Sap1-10) plays a critical role in the pathogenicity of Candida. Considering the increasing prevalence rate of candidiasis worldwide, understanding of Sap family’s pathogenicity mechanism becomes critical for the development of any new antifungal agents or therapies. This study mainly investigated the virulent mechanisms of Sap7 and Sap9 in Candida infection, as well as the synergistic effect of HIV protease inhibitors and fluconazole on antifungal treatment. The first part of this study was to investigate the role of C. albicans SAP7 in fungal invasion to oral epithelial cells. sap7Δ/Δ mutant strain was created using a PCR-based gene disruption method via homologous recombination. Compared the wild type strain with sap7Δ/Δ mutant strain, the results demonstrated that the deletion of SAP7 restrained C. albicans invasion to oral epithelial cells. C. albicans sap7Δ/Δ mutant strain significantly reduced the induced endocytosis by 47.8%. Approximate 25% attenuation of sap7Δ/Δ mutant strain was internalized into the oral epithelial cells, and 27% reduction of the release of lactate dehydrogenase was detected. Evaluation of reconstituted human epithelial (RHE) tissues showed a significant reduction in the number of attached and invaded fungal cells to oral epithelial tissues compared with the wild type strain. The data implied that SAP7 contributes to C. albicans invasion to oral epithelial cells. The second part of the study was to investigate the role of C. albicans SAP9 in hyphae formation and invasion ability. Hyphal development was examined by both liquid and solid hyphal inducing media. Compared the sap9Δ/Δ mutant strain with wild type strain, hyphal formation tests demonstrated that the yeast-to-hyphal morphological transition was deficient in the sap9Δ/Δ mutant strain. Besides, quantitative reverse transcription-PCR (qRT-PCR) revealed a significant down regulation of the expression of EFG1 (approximately 40%) in sap9Δ/Δ mutant strain. EFG1 is a transcription factor gene positively mediating hyphae formation in C. albicans. It was also found that approximately 70% reduced sap9Δ/Δ mutant strain was endocytosed by host cells and 40% attenuation of host cell damage caused by sap9Δ/Δ mutant strain, compared with the wild type strains. The data strongly suggested that C. albicans Sap9 is a novel hyphal associated factor via the cAMP-protein kinase A pathway, leading to invasion into the host cells and causing cell damage. The final part of the study was to investigate the synergistic antifungal effect of HIV protease inhibitors and fluconazole on C. albicans. The results showed that while the MIC of saquinavir and fluconazole required to suppress the candidal growth was 700 μM and 32.64μM respectively, the combination of saquinavir (12.5 μM) and fluconazole (4.08 μM) at lower concentrations could act synergistically against fungal growth. SAP1-10 mRNA expressions are significantly less using combined treatment than fluconazole alone. Our data indicated that combined therapy might be an alternative way to reduce the dosage of antifungal drug and minimize multidrug resistance.published_or_final_versionDentistryDoctoralDoctor of Philosoph