Role of Cryptococcus neoformans Rho1 GTPases in the PKC1 Signaling Pathway in Response to Thermal Stress

Abstract

ABSTRACTTo initiate and establish infection in mammals, the opportunistic fungal pathogenCryptococcus neoformansmust survive and thrive upon subjection to host temperature. Primary maintenance of cell integrity is controlled through the protein kinase C1 (PKC1) signaling pathway, which is regulated by a Rho1 GTPase inSaccharomyces cerevisiae. We identified threeC. neoformansRho GTPases, Rho1, Rho10, and Rho11, and have begun to elucidate their role in growth and activation of the PKC1 pathway in response to thermal stress. Western blot analysis revealed that heat shock of wild-type cells resulted in phosphorylation of Mpk1 mitogen-activated protein kinase (MAPK). Constitutive activation of Rho1 caused phosphorylation of Mpk1 independent of temperature, indicating its role in pathway regulation. A strain with a deletion ofRHO10also displayed this constitutive Mpk1 phosphorylation phenotype, while one with a deletion ofRHO11yielded phosphorylation similar to that of wild type. Surprisingly, like arho10Δ strain, a strain with a deletion of bothRHO10andRHO11displayed temperature sensitivity but mimicked wild-type phosphorylation, which suggests that Rho10 and Rho11 have coordinately regulated functions. Heat shock-induced Mpk1 phosphorylation also required the PKC1 pathway kinases Bck1 and Mkk2. However, Pkc1, thought to be the major regulatory kinase of the cell integrity pathway, was dispensable for this response. Together, our results argue that Rho proteins likely interact via downstream components of the PKC1 pathway or by alternative pathways to activate the cell integrity pathway inC. neoformans.