Analyses of Msb2, Mp65 and Wsc1 in the cell wall integrity pathway and cell-cell signaling in Candida albicans biofilms.

摘要

As a distinguishing structure novel to fungal cells, the cell wall is an important therapeutic target. Therefore, the understanding of mechanisms and pathways that determine cell wall dynamics are relevant to fungal pathogenesis. Our focus in the study is the cell wall of Candida albicans, a major invasive fungal pathogen of humans. The ability of C. albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. There are few studies in C. albicans that describe any upstream cell wall sensor that serves as an adapter between the cell wall damage inputs and the downstream cell wall damage response cascade. Here we investigate the role of three candidate cell surface proteins - Msb2, Mp65 and Wsc1 as sensors of cell wall damage response in C. albicans. Our reason for selecting these as candidate genes stems from the finding that mutant strains of each of these three cell wall genes are sensitive to caspofungin and we wanted to test their role as upstream/cell surface sensors for known cell wall stress response genes. We find that mutant strains for these genes are sensitive to caspofungin and complementation with a wild type copy restores the mutant phenotype. However, there is no apparent alteration in transcriptional response in these mutants for the selected candidate cell wall integrity genes; suggesting the existence of other altered pathways.;Biofilms of Candida albicans include both yeast cells and hyphae. Prior studies indicate that a zap1Delta/Delta mutant, defective in zinc regulator Zap1, has increased accumulation of yeast cells in biofilms. This altered yeast-hypha balance may arise from internal regulatory alterations, or from an effect on production of diffusible quorum-sensing (QS) molecules. Here we develop biosensor reporter strains that express yeast-specific YWP1-RFP or hypha-specific HWP1-RFP, along with a constitutive TDH3-GFP normalization standard. Seeding these biosensor strains into biofilms allows a biological activity assay of the surrounding biofilm milieu. A zap1Delta/Delta biofilm induces the yeast-specific YWP1-RFP reporter in a wild-type biosensor strain, as determined by both QRTPCR gene expression measurements and confocal microscopy. Remediation of the zap1Delta/Delta zinc uptake defect through zinc transporter gene ZRT2 overexpression reverses induction of the yeast-specific YWP1-RFP reporter. GC-MS measurements of known organic QS molecules show that the zap1Delta/Delta mutant accumulates significantly less farnesol than wild-type or complemented strains, and that ZRT2 overexpression does not affect farnesol accumulation. Farnesol is a well characterized inhibitor of hypha formation; hence a reduction in farnesol levels in zap1Delta/Delta biofilms is unexpected. Our findings argue that a Zap1- and zinc-dependent signal affect the yeast-hypha balance and is operative in the low-farnesol environment of the zap1Delta/Delta biofilm. In addition, our results indicate that Zap1 is a positive regulator of farnesol accumulation.