Candidaalbicansmutants with mutations in mitogen-activated protein (MAP) kinaseHOG1displayed 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.albicansHog1 was activated not only in response to an increase in external osmolarity, as happens with itsSaccharomycescerevisiaehomologue, but also in response to hydrogen peroxide. The Hog1-mediated response to oxidative stress was different from that of transcription factor Cap1, the homologue ofS.cerevisiaeYap1, as shown by the different sensitivities to oxidants and the kinetics of cell death ofcap1Δ,hog1, andhog1cap1Δ mutants. Deletion ofCAP1did not influence the level of Hog1 phosphorylation, and deletion ofHOG1did not affect Cap1 nuclear localization. Moreover, we show that theHOG1gene plays a role in chlamydospore formation, another oxygen-related morphogenetic event, as demonstrated by the fact thathog1cells were unable to generate these thick-walled structures in several media through a mechanism different from that of theEFG1regulator. 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 inC.albicans.

MAP (mitogen-activated protein) kinase-mediated pathways are key elements in sensing and transmitting the response of cells to environmental conditions by the sequential action of phosphorylation events. In the fungal pathogenCandida albicans, different routes have been identified by genetic analysis, and especially by the phenotypic characterization of mutants altered in the Mkc1, Cek1/2 and Hog1 MAP kinases. The cell integrity (orMKC1-mediated) pathway is primarily involved in the biogenesis of the cell wall. The HOG pathway participates in the response to osmotic stress while the Cek1 pathway mediates mating and filamentation. Their actual functions are, however, much broader and Mkc1 senses several types of stress, while Hog1 is also responsive to other stress conditions and participates in two morphogenetic programmes: filamentation and chlamydospore formation. Furthermore, it has been recently shown that Cek1 participates in a putative pathway involved in the construction of the cell wall and which seems to be operative under basal conditions. As these stimuli are frequently encountered in the human host, they provide a reasonable explanation for the significant reduction in pathogenicity that several signal transduction mutants show in certain animal models of virulence. MAPK pathways therefore represent an attractive multienzymic system for which novel antifungal therapy could be designed.

Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species ( Candida albicans , Candida parapsilosis , Candida glabrata , Candida tropicalis , Candida krusei , Cryptococcus neoformans , and Cryptococcus gattii ). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.

The Sho1 adaptor protein is an important element of one of the two upstream branches of the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. In the present work, we describe its role in the pathogenic yeast Candida albicans by the construction of mutants altered in this gene. We report here that sho1 mutants are sensitive to oxidative stress but that Sho1 has a minor role in the transmission of the phosphorylation signal to the Hog1 MAP kinase in response to oxidative stress, which mainly occurs through a putative Sln1-Ssk1 branch of the HOG pathway. Genetic analysis revealed that double ssk1 sho1 mutants were still able to grow on high-osmolarity media and activate Hog1 in response to this stress, indicating the existence of alternative inputs of the pathway. We also demonstrate that the Cek1 MAP kinase is constitutively active in hog1 and ssk1 mutants, a phenotypic trait that correlates with their resistance to the cell wall inhibitor Congo red, and that Sho1 is essential for the activation of the Cek1 MAP kinase under different conditions that require active cell growth and/or cell wall remodeling, such as the resumption of growth upon exit from the stationary phase. sho1 mutants are also sensitive to certain cell wall interfering compounds (Congo red, calcofluor white), presenting an altered cell wall structure (as shown by the ability to aggregate), and are defective in morphogenesis on different media, such as SLAD and Spider, that stimulate hyphal growth. These results reveal a role for the Sho1 protein in linking oxidative stress, cell wall biogenesis, and morphogenesis in this important human fungal pathogen.

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.