The Tricalbin-Family Endoplasmic Reticulum-Plasma Membrane Tethering Proteins Attenuate ROS-Involved Caspofungin Sensitivity in Candida albicans

The endoplasmic reticulum-plasma membrane (ER-PM) contacts are one kind of important membrane contact structures in eukaryotic cells, which mediate material and message exchange between the ER and the PM. However, the specific types and functions of ER-PM tethering proteins are poorly understood in the human fungal pathogen Candida albicans. In this study, we observed that the two tricalbin-family proteins, i.e., Tcb1 and Tcb3, were colocalized with the ER-PM contacts in C. albicans. Deletion of the tricalbin-encoding genes TCB1 and TCB3 remarkably reduced ER-PM contacts, suggesting that tricalbins are ER-PM tethering proteins of C. albicans. Stress sensitivity assays showed that the TCB-deleted strains, including tcb1Δ/Δ, tcb3Δ/Δ, and tcb1Δ/Δ tcb3Δ/Δ, exhibited hypersensitivity to cell wall stress induced by caspofungin. Further investigation revealed that caspofungin induced drastic reactive oxygen species (ROS) accumulation in the mutants, which was attributed to enhanced oxidation of Ero1 in the ER lumen. Removal of intracellular ROS by the ROS scavenger vitamin C rescued the growth of the mutants under caspofungin treatment, indicating that Ero1 oxidation-related ROS accumulation was involved in caspofungin hypersensitivity of the mutants. Moreover, deletion of the TCB genes decreased secretion of extracellular aspartyl proteinases, reduced transport of the cell wall protein Hwp1 from the cytoplasm to the cell wall, and attenuated virulence of the fungal pathogen. This study sheds a light on the role of ER-PM tethering proteins in maintenance of cell wall integrity and virulence in fungal pathogens. IMPORTANCE The endoplasmic reticulum-plasma membrane contacts are important membrane contact structures in eukaryotic cells, functioning in material and message exchange between the ER and the PM. We observed that the two tricalbin-family endoplasmic reticulum-plasma membrane contact proteins are required for tolerance to caspofungin-induced cell wall stress in the pathogenic fungus Candida albicans. The tricalbin mutants exhibited hypersensitivity to cell wall stress induced by caspofungin. Further investigation revealed that Ero1 oxidation-related reactive species oxygen accumulation was involved in caspofungin hypersensitivity of the tricalbin mutants. Moreover, loss of tricalbins reduced secretion of extracellular aspartyl proteinases, decreased transport of the cell wall proteins from the cytoplasm to the cell wall, and attenuated virulence of the fungal pathogen. This study uncovers the role of ER-PM tethering proteins in sustaining protein secretion, maintenance of cell wall integrity and virulence in fungal pathogens.