The Hog1 mitogen-activated protein kinase is essential in the oxidative stress response and chlamydospore formation in Candida albicans

Abstract

Candida albicans mutants with mutations in mitogen-activated protein (MAP) kinase HOG1 displayed an increased sensitivity to agents producing reactive oxygen species, such as oxidants (menadione, hydrogen peroxide, or potassium superoxide), and UV light. Consistent with this finding, C. albicans Hog1 was activated not only in response to an increase in external osmolarity, as happens with its Saccharomyces cerevisiae homologue, but also in response to hydrogen peroxide. The Hog1-mediated response to oxidative stress was different from that of transcription factor Cap1, the homologue of S. cerevisiae Yap1, as shown by the different sensitivities to oxidants and the kinetics of cell death of cap1Delta, hog1, and hog1 cap1Delta mutants. Deletion of CAP1 did not influence the level of Hog1 phosphorylation, and deletion of HOG1 did not affect Cap1 nuclear localization. Moreover, we show that the HOG1 gene plays a role in chlamydospore formation, another oxygen-related morphogenetic event, as demonstrated by the fact that hog1 cells were unable to generate these thick-walled structures in several media through a mechanism different from that of the EFG1 regulator. This is the first demonstration of the role of the Hog1-mediated MAP kinase pathway in resistance to oxidative stress in pathogenic fungi, and it allows us to propose a molecular model for the oxidative stress response in C. albicans.

FIG. 1.

C. albicans hog1 mutants are sensitive to oxidative stress. (A) C. albicans cells were exposed to 100 mM hydrogen peroxide, 40 mM MD, and 2 mM KO₂ in liquid YPD medium. Samples were taken at different times, diluted, and spotted onto YPD plates (10⁵ cells in 10 μl). (B) A total of 100 to 200 C. albicans cells were plated on YPD plates and irradiated at 254 nm at different times by using a UV source. Plates were incubated at 37°C, and the percent survival was calculated by CFU counting after 24 h. The results are the means of three experiments. The standard error was always less than 10%. Strains used were as follows: Wt, HOG1/HOG1; H/h, HOG1/hog1; h/h, hog1/hog1; and h/h + H, HOG1 reintegrated in a hog1/hog1 strain (Table 1).

FIG. 2.

Hog1 is phosphorylated in the presence of NaCl and H₂O₂ with similar kinetics. Western blots show the activation of Hog1 after H₂O₂ (10 mM) or NaCl (1.2 M) treatment for 10 min (A) or in a kinetic study at different times (B). Intermediate times were analyzed in the kinetic study, but only representative time points are shown. The same blots were assayed with a monoclonal antibody raised against phospho-p38 (Anti-TGY^P) and a polyclonal antibody raised against S. cerevisiae Hog1 (Anti-ScHog1) (see Materials and Methods). See the legend to Fig. 1 for strain designations.

FIG. 3.

hog1 cap1Δ knockout strains are more sensitive to oxidative stress than hog1 or cap1Δ knockout strains. (A) C. albicans exponentially growing cells were exposed to 50 mM hydrogen peroxide in liquid YPD medium as indicated in Materials and Methods and spotted onto YPD plates. The plates were incubated at 37°C for 24 h. (B) The viability of different strains in YPD medium containing 100 mM hydrogen peroxide was determined by fluorescence-activated cell sorting analysis with propidium iodide as a cell death marker. Error bars indicate standard errors. c/c, cap1Δ/cap1Δ strain; h/h c/c, hog1/hog1 cap1Δ/cap1Δ strain; see the legend to Fig. 1 for other strain designations.

FIG. 4.

hog1 and cap1Δ knockout strains behave differently, the hog1 cap1Δ double mutant having some mixed phenotypes. (A) Responses to high osmolarity and heavy metal stress of wild-type and mutant strains. Serial 10-fold dilutions of exponentially growing cells of the strains indicated were spotted onto YPD plates containing 1 M NaCl or 150 μM CdSO₄. (B) Responses to different types of oxidative stress of wild-type and mutant strains. Serial 10-fold dilutions of exponentially growing cells of the strains indicated were spotted onto YPD plates containing 5 mM H₂O₂ or 300 μM MD. (C) Catalase activities in hog1, cap1Δ, and hog1 cap1Δ mutants. The results are the means and standard deviations of three experiments.

FIG. 5.

Upon oxidative stress, Cap1 did not affect the phosphorylation of Hog1, and Hog1 did not affect the nuclear localization of Cap1. (A) Hog1 is phosphorylated in the presence of hydrogen peroxide independently of Cap1. A Western blot shows the Cap1-independent activation of Hog1 after 1 h in the presence of 10 mM H₂O₂. The same blot was assayed with a monoclonal antibody raised against phospho-p38 (Anti-TGY^P) and a polyclonal antibody raised against S. cerevisiae Hog1 (Anti-ScHog1) (see Materials and Methods). (B) Cap1 localization is not affected in a hog1 mutant upon oxidative stress. Cap1 localizes to the nucleus in the presence of oxidative stress (0.4 mM H₂O₂, 15 min) irrespective of the genetic background. Cells were observed by phase-contrast microscopy (PC), and nuclei were observed by using propidium iodide (IP) to ascertain the localization of the Cap1-GFP fusion (GFP). More than 300 cells were observed, and representative images of different strains were chosen.

FIG. 6.

hog1 mutants show defects in chlamydospore formation. Images show the wild-type strain (A and B) and hog1 mutants (C) on corn meal agar. No formation of chlamydospores can be observed at the end of the hog1 mutant hyphae, in contrast to those of the wild-type strain.

FIG. 7.

C. albicans seems to respond to oxidative stress in a manner different from those of S. cerevisiae and S. pombe. The scheme shows the different ways in which C. albicans, S. cerevisiae, and S. pombe respond to oxidative stress. N, nucleus. The white bar inside the nucleus represents DNA. See Discussion for a detailed explanation.