Regulation of Phosphatidylinositol-(4,5)-bisphosphate and Active-Rho1p Levels and Distribution is Crucial for Correct Spatio-temporal Cytokinesis and Echinocandin Responses in Candida albicans

Abstract

Candida species cause severe infections like invasive candidiasis, which annually affect 1.5 million people worldwide and cause close to 1 million deaths. Candida albicans is the predominant cause of candidiasis. We previously showed that EH domain-containing protein Irs4p binds 5-phosphatase enzyme Inp51p to regulate plasma membrane levels of phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) in C. albicans. Indeed, deletion of IRS4 or INP51 led to elevated levels of PI(4,5)P2 and presence of abnormal intracellular membranous PI(4,5)P2 patches. We demonstrated an interplay between PI(4,5)P2 and septins to regulate the PKC-Mkc1 cell wall integrity pathway, echinocandin and cell wall stress responses, and virulence during candidiasis. In the current investigation, we used fluorescent protein tagging and live cell imaging to follow the nascency of PI(4,5)P2 patches. We show that these abnormal patches tightly correlate with cytokinesis, as they predominantly arise close to the site and time of cell division. We further demonstrate these patches colocalize PI(4,5)P2 with actomyosin ring components Act1p and Myo1p, which form its core, and active Rho1p, a small GTPase that plays a regulatory role. Additionally, activation of Rho1p was altered in irs4 and inp51 mutants compared to wild-type strain, with over-activation or down-activation during early exponential or stationary phase, respectively. Wild-type cells exposed to 4xMIC of the echinocandin caspofungin show abnormal PI(4,5)P2 patches colocalizing the same cytokinesis components as above, except that they were transient. Taken together, our results support a model in which PI(4,5)P2 plays a pivotal role, along with Rho1p, in the correct execution of cytokinesis and response to caspofungin.