MAPKKK-independent Regulation of the Hog1 Stress-activated Protein Kinase in Candida albicans

Jill Cheetham,Donna M Maccallum,Kathryn S Doris,Alessandra Da Silva Dantas,Susan Scorfield,Frank Odds,Deborah A Smith,Janet Quinn

doi:10.1074/jbc.m111.265231

Jill Cheetham, Donna M Maccallum + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m111.265231

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Abstract

The Hog1 stress-activated protein kinase 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 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable 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 previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2DD cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression 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.

Highlights

Stress responses are intimately linked with the pathogenicity of the dimorphic fungus Candida albicans, the major systemic fungal pathogen of humans
Mutation of the consensus phosphorylation sites of the C. albicans Hog1 antibody (Hog1) stress-activated protein kinase (SAPK) and the Pbs2 MAPKK to nonphosphorylatable residues resulted in strains that displayed equivalent stress-sensitive phenotypes to those exhibited by hog1⌬ and pbs2⌬ null mutants (Figs. 1 and 2)
This is reminiscent of studies in S. cerevisiae in which it was shown that Hog1 requires the conserved phosphorylation sites to prevent cross-talk to the Cek1-related Kss1/Fus3 MAPKs [27]

Summary

Strains used in this study

C. albicans RM1000 BWP17 JC21 JC64 JC50 JC52 JC36 JC154 JC76 JC80 JC72 JC74 JC112 JC124 JC126 JC167 JC174 JC181 JC482 JC507 JC517 JC1614 JC1616 JC1618 JC1620. Expression of S. cerevisiae Pbs or S. pombe Wis mutants, in which the predicted phosphorylation sites were mutated to the phosphomimetic aspartic acid residue, resulted in constitutive activation of the Hog and Sty SAPKs, respectively [28, 29]. This proved lethal in S. cerevisiae, subsequent analysis of S. pombe wis1-DD cells revealed a novel MAPKKK-independent mechanism of SAPK activation in response to heat stress [28, 30]. Do our data uncover mechanisms of SAPK regulation in C. albicans not previously documented in S. cerevisiae and S. pombe, but they demonstrate that the attenuated virulence exhibited by hog1⌬ cells is likely attributed to impaired stress responses rather than defects in morphogenesis

EXPERIMENTAL PROCEDURES

Oligonucleotides used in this study

RESULTS

Outcome score

DISCUSSION