Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans

Abstract

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.

FIG. 1

Terminal phenotype of hog1 mutants. (A) Effect of osmostress (1.0 M NaCl) on the growth of wild-type cells (SC5314) or the hog1 mutant (CNC13) in liquid YED medium. The OD (estimated as the A₆₀₀) is plotted versus time. The y-axis scale is logarithmic. (B) Propidium iodide-stained cells of the hog1 mutant after 24 h of growth under restrictive conditions (right panel). Arrows indicate dead cells in the clusters observed under phase-contrast microscopy (left panel). (C) Flow cytometric analysis of the DNA content of mutant cultures grown in parallel on YED medium or YED medium plus 1 M NaCl. The peaks observed for control cells (labeled YED 0′) represent 2n (left) and 4n (right) DNA content, while the numbers indicate the time in minutes after the transfer to the restrictive conditions. (D) Microscopic analysis of hog1 mutant cells under restrictive conditions (left panel; phase-contrast image), showing nuclei (right panel; fluorescence image). The arrow indicates a detail of a nucleus in the process of segregation to the new bud. A representative cluster of cells is shown. Bars, 1 μm.

FIG. 2

Scanning and transmission electron microscopy of hog1 mutants. (A) Scanning electron microscopy of hog1 mutant (CNC13) (left panel) or wild-type (SC5314) (right panel) cells after 24 h of growth under restrictive conditions. The arrow indicates a characteristic cell with a symmetric type of division. (B) Transmission electron microscopy of similar samples. Arrows indicate almost completely separated but still connected cells with well-formed septa. Bars, 1 μm.

FIG. 3

Defects in bud site selection after osmotic shock. Time lapse photography of hog1 mutant cells under solid YPD medium supplemented with 0.75 M NaCl. Numbers indicate the time (in hours) after the transfer to restrictive conditions. Arrows labeled “a” indicate small buds, while the arrow labeled “b” indicates a newly formed bud. Bars, 1 μm.

FIG. 4

Antifungal susceptibility and cell wall architecture. (A) Different amounts of cells (indicated at the top of the rows) from the indicated C. albicans strains were spotted onto YEPD medium (as a control) or YEPD medium supplemented with Congo red at 150 μg/ml and incubated at 37°C. (B) Experiments similar to those described for panel A were done with the S. cerevisiae strains indicated; cells were spotted onto YEPD medium supplemented with a 150-μg/ml final concentration of calcofluor white and incubated at 30°C for 24 h. (C) Chitinase activity in cell extracts after 24 and 48 h of growth under nonrestrictive (1 M NaCl) (left panel) and restrictive (right panel) conditions. Units of activity (UA) (see Material and Methods) per milligram of dried extract are given in the y axis. Data are the mean value of two independent experiments.

FIG. 5

Colony morphology of hog1 mutants. Colony morphology of wild-type (SC5314) and mutant hog1 (CNC13) cells on different solid media. Approximately 50 CFU were spread onto either YED medium, Spider medium, minimal SD medium plus 10% bovine fetal serum, or SLAD medium on petri dishes and incubated for 7 days at 37°C before photographs were taken. The colony borders are shown for cells on Spider medium.

FIG. 6

Effect of Hog1p on the serum-induced dimorphic transition. (A) Cells from the indicated strains were inoculated in YED medium plus bovine fetal serum at different concentrations (20, 10, 5, and 1%), and phase-contrast microphotographs were taken after 6 h of incubation at 37°C. (B) Border colony morphologies of S. cerevisiae L5366 transformed with vector YEp352 (left picture) or the multicopy plasmid pHOG1c24.2 (bearing the C. albicans HOG1 gene) (right picture) are shown. Cells were plated onto SLAD medium, and pictures were taken after growth for 6 days at 30°C. Bars, 10 μm.

FIG. 7

Virulence assays. Standard survival curves of BALB/c mice infected systemically with 10⁶ (A) or 10⁷ (B) cells of the C. albicans strains indicated in the figure. Since strains CNC15-10, RM100, and CNCH1 at a dose of 10⁷ gave curves similar to the one shown for SC5314 in panel B, these results are therefore not shown for clarity.