MAPKKK-Independent regulation of the Hog1 stress activated protein kinase in Candida albicans

The Hog1 stress activated protein kinase (SAPK) regulates both stress responses and morphogenesis in Candida albicans, and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr174/Tyr176) and Pbs2 (Ser355/Thr359), to non-phosphorylatable residues, resulted in strains that phenocopied hog1¿ and pbs2¿ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing non-phosphorylatable Pbs2 (Pbs2AA). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2DD) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2DD cells. Collectively, these data reveal a hitherto uncharacterised MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2DD cells did not exhibit high basal levels of Hog1 phosphorylation, over-expression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation which was further increased upon stress. Significantly, both Pbs2AA and Pbs2DD cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2AA cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.