Ssk1p ofCandida albicansis a putative response regulator protein of the Hog1 two-component signal transduction system. InSaccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown thatC. albicans SSK1does not complement thessk1mutant ofS. cerevisiaeand that thessk1mutant ofC. albicansis not sensitive to sorbitol. In this study, we show that theC. albicans ssk1mutant is sensitive to several oxidants, including hydrogen peroxide,t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by thessk1mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30°C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). InS. cerevisiae, three (AHP1,HSP12, andPYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis inC. albicans. Of the down-regulated genes,ALS1is a known cell adhesin inC. albicans. Verification of the microarray data was obtained by reverse transcription-PCR forHSP12,AHP1,CHK1,PYC2,GPH1,ALS1,MNN4-4, andFLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway inC. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in thessk1mutant ofC. albicanswhen grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in thessk1mutant ofC. albicanswhen grown in a hyperosmotic medium but was not phosphorylated in thessk1mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate thatC. albicansutilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further,SSK1appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions ofC. albicans SSK1differ from those ofS. cerevisiae SSK1.

The Hog1 MAP kinase pathway regulates stress adaptation in several fungi. To assess its role in stress adaptation in Aspergillus fumigatus, we constructed mutants in genes encoding the sensor histidine kinase (HK) tcsB as well as sakA, which are homologues of the Saccharomyces cerevisiae sln1 and Hog1, respectively. Compared to the wild type strain (Wt), growth of sakA (sakA▵) mutant was reduced, and growth inhibition was increased when H₂O₂, menadione, or SDS was added to the media. On the other hand, the tcsB mutant (tcsB▵) was similar to the Wt strain in regard to growth and morphology, although a partial sensitivity to SDS was observed. Western blot analysis of Wt and the tcsB▵ strains indicated that when stressed with H₂O₂, phosphorylation of Hog1p still occurs in the mutant. Since in Candida albicans, Hog1 regulates transcription of at least one histidine kinase, we performed RT-PCR of 6 histidine kinase genes as well as the ssk1 and skn7 response regulator genes of A. fumigatus. No significant differences in transcription were observed with the sakA▵ when compared to the Wt, indicating that the sakA does not regulate transcription of these genes. Our studies indicate that the A. fumigatus sakA is required for optimal growth of the organism with or without oxidant stress, while tcsB gene is dispensable.

Using a Tn 7 transposon library of Candida albicans , we have identified a mutant that exhibited sensitivity in drop plate assays to oxidants such as menadione and hydrogen peroxide. To verify the role of the mutated gene in stress adaptation, null mutants were constructed and phenotypically characterized. Because of its apparent functions in growth and oxidant adaptation, we have named the gene GOA1 . Goa1p appears to be unique to the CTG subclade of the Saccharomycotina , including C. albicans . Mutants of C. albicans lacking goa1 (strain GOA31) were more sensitive to 6 mM H ₂ O ₂ and 0.125 mM menadione than the wild type (wt) or a gene-reconstituted (GOA32) strain. The sensitivity to oxidants correlated with reduced survival of the GOA31 mutant in human neutrophils and avirulence compared to control strains. Other phenotypes of GOA31 include reduced growth and filamentation in 10% serum, Spider, and SLAD agar media and an inability to form chlamydospores. Since Goa1p has an N-terminal mitochondrion localization site, we also show that green fluorescent protein-tagged Goa1p shows a mitochondrionlike distribution during oxidant or osmotic stress. Further, the inability of GOA31 to grow in medium containing lactate, ethanol, or glycerol as the sole carbon source indicates that the mitochondria are defective in the mutant. To determine how Goa1p contributes to mitochondrial function, we compared the wt, GOA32, and GOA31 strains for mitochondrial electrical membrane potential, respiration, and oxidative phosphorylation. We now show that GOA31, but not the wt or GOA32, had decreased respiration and mitochondrial membrane potential such that mutant cells are unable to drive oxidative phosphorylation. This is the first report in C. albicans of a respiratory defect caused by a loss of mitochondrial membrane potential.

New data suggest that the global incidence of several types of fungal diseases have traditionally been under-documented. Of these, mortality caused by invasive fungal infections remains ...

The SKN7 two-component response regulator gene of Candida albicans was deleted, and the phenotype of the mutant was established. This mutant exhibited impaired growth on Spider agar and 10% serum agar compared to wild-type and gene-reconstituted strains. The skn7 mutant was sensitive to H ₂ O ₂ in vitro, but its virulence was only mildly attenuated. A comparison of the Skn7p and Ssk1p response regulators of C. albicans is discussed.