Abstract
ABSTRACT
Ssk1p of
Candida albicans
is a putative response regulator protein of the Hog1 two-component signal transduction system. In
Saccharomyces cerevisiae
, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that
C. albicans SSK1
does not complement the
ssk1
mutant of
S. cerevisiae
and that the
ssk1
mutant of
C. albicans
is not sensitive to sorbitol. In this study, we show that the
C. albicans ssk1
mutant 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 the
ssk1
mutation. 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). In
S. cerevisiae
, three (
AHP1
,
HSP12
, and
PYC2
) 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 in
C. albicans
. Of the down-regulated genes,
ALS1
is a known cell adhesin in
C. albicans
. Verification of the microarray data was obtained by reverse transcription-PCR for
HSP12
,
AHP1
,
CHK1
,
PYC2
,
GPH1
,
ALS1
,
MNN4
-
4
, and
FLO1
. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in
C. albicans
, we used Western blot analysis to measure phosphorylation of Hog1p in the
ssk1
mutant of
C. albicans
when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the
ssk1
mutant of
C. albicans
when grown in a hyperosmotic medium but was not phosphorylated in the
ssk1
mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that
C. albicans
utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further,
SSK1
appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of
C. albicans SSK1
differ from those of
S. cerevisiae SSK1
.