Identification And Functional Study Of Sac1 In Candida Albicans

Candida albicans is one of the most important opportunistic fungal pathogens in clinical practice. Candida albicans infections, especially systemic infections, are urgent due to the increase of immunocompromised individuals. During the interaction of C. albicans with the host, this pathogen evolved elaborate systems functioning in morphogenesis and stress response, which are important for efficient colonization and survival in host immune attacks. Phosphatidyl inositol phosphates (PIs) are one type of important small molecules involved in signal transduction and regulation of growth and metabolism. In Saccharomyces cerevisiae and mammalian cells, the phosphatidyl inositol phosphatase Sacl and its homologs mediate the transmission of PIP and PIP2 to PI, functioning in organization of actin skeleton, determination of cell polarization, maintenance of cell wall integrity (CWI), endocytosis and exocytosis. However, the characterization and function of Sacl in C. albicans remains to be investigated. This study aims to identify Sacl and to explore its function in this pathogen. The main results are demonstrated as follows.(1) BLASTP analysis revealed a homolog of ScSacl in Candida albicans genome database, named Sacl. Sequence analysis showed that this protein is a member of proteins containing SAC domain, which is characterized by a conserved CXsR(T/S) motif. Complementatin tests found that SAC1 can rescue the growth defect in inositol metabolism of ScsaclA. Sacl-GFP localization revealed that this protein is an endoplasmic reticulum (ER)-localized membrane protein.(2) Using two-step PCR mediated homogenous recombination and the integration system, SAC1 deletion strain and its reconstituted strain of C. albicans were constructed. Solid dotting experiments showed that deletion of SAC1 caused high-sensitivity to ER stress and cell wall stress. Acid phosphatase assays showed that this deletion led to a defect in glycosylation. Chitin staining and transmission electron microscopy revealed that this deletion caused abnormal distribution and increased content of cell wall chitin, and abnormal cell wall ultramicroscopic structure. GFP reporting systems and real-time PCR assays further demonstrated that SAC1 deletion up-regulated the expression of unfolded protein response (UPR) genes and cell wall integrity (CWI) genes. Hence, Sacl has an important role in maintenance of both ER function and CWI.(3) Deletion of SAC1 caused decreased ability of hyphal development, and the mutant formed short and curved hyphae, suggesting that the mutant had a defect in polarized growth. Actin staining and Hwpl localization revealed that this deletion led to abnormal localization of actin patches at the hyphal tip, and a defect in polarized transport of Hwp1, indicating that Sacl regulates polarized transport of hypha-specific proteins by maintaining the tip localization of actin cytoskeleton.(4) Deletion of SAC] led to significant decrease in resistance ability against macrophage attacks, and in invasion of host epithelial cells and systemic infection to mice, suggesting that Sacl is involved in C. albicans pathogenicity, and is an impotant virulence-related factor.