Our group is interested in the role of MAPK (Mitogen Activated Protein Kinases) pathways in lower eukaryotes such as Saccharomyces cerevisiae and the opportunistic fungus Candida albicans. We have shown that Hog1 is a virulence determinant in C. albicans, as shown by the reduced virulence of strains deleted in this gene in an experimental infection model (Alonso Monge, 1999). The approach of this study is the use of S. cerevisiae as a tool to analyse the expression of human genes related to fungal HOG1, the MAPK Hog1p involved in the High Osmolarity Glycerol response (HOG pathway). We have expressed and analysed the human genes JNK1, p38˜and ERK5 in S. cerevisiae with the aim of designing specific screening for inhibitors of this potential target in antifungal therapy. JNK1 belongs to the JNK (c-Jun NH2 Kinases) human MAPK family, a SAPK (Stress-activated Protein Kinases) type kinase, as shown by the activation of this MAPKs by environmental stresses (heat shock, oxidative stress and ionizing radiations among others) and inflammatory citokines of the TNF (Tumor Necrosis Factor). It has been described that JNK1 complements the osmosensitivity of a S. cerevisiae hog1 •mutant (Galcheva-Gargova et al., 1994). Another member of the human MAPK family is p38 ••a member of the p38 family; this family is activated by similar stimuli that recruit JNKs and are considered a second group of SAPKs. p38 •is the most similar (52% of identity) human MAPK respect to S. cerevisiaeHog1p. p38 (and p38 are targets for the experimental anti-inflamatory CSAIDs•Citokine-suppressive Anti-Inflammatory Drugs). p38 •complements the osmosensitivity of a S. cerevisiae hog1 ••mutant (Han et al., 1994). Finally, ERK5 (Extracellular-signal Regulated Kinase) is a third class of SAPK. It has been described that this kinase responds to both oxidative and osmotic stresses (Abe et al., 1996). The goal of this work is the functional comparison of these human SAPKs with fungal Hog1p, using S. cerevisiae as a heterologous expression system. For this purpose, we have analized the complementation of a S. cerevisiae hog1 ••mutant sensitivity to both osmotic and oxidative stresses. We have carried out western-blottings assays in order to study the phosphorylation of these proteins showing that S. cerevisiae can only phosphorylate them in response to osmotic stress but not to oxidative stress. Morphogenetic defects of the hog1 •mutants under hyperosmotic stress are only suppressed by fungal Hog1p. Our data demonstrate the functional conservation of MAPKs although there are differential aspects among the three human SAPKs and functional differences between the group of human SAPKs and fungal MAPKs.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)