Le rôle de RIM101p dans la réponse au pH chez Candida albicans

Rim101p is a conserved fungal transcription factor that becomes activated through C-terminal cleavage at alkaline pH. This work describes the identification and analysis of Rim101p targets in Candida albicans. A constitutively active truncated version of Rim101p was introduced under the control of the MET3-promotor into a rim101 null mutant to monitor Rim101-dependent transcriptional changes. Transcriptional changes were recorded using microarrays along a time course following induction of RIM101SL transcription. The transcriptional patterns of the 133 regulated genes were clustered into five classes. Analysis of their promoters showed an enrichment of putative Rim101p binding and permitted to identify an extended Rim101p binding motif. Microarray results were confirmed on 20 selected genes by quantitative real-time PCR. Furthermore, the relevance of the microarray data for the pH response of C. Albicans was assessed by monitoring transcriptional changes of these genes in a wild type strain grown at pH 4 or pH 8. In spite of these experimental setup differences, a clear correlation of the results was observed for a large majority of the tested genes. Microarray data suggested that Rim101p activity had a strong impact on the expression of genes of the ALS (Agglutinin-Like Sequence) gene family. The extremely high sequence conservation within this family hampered however a gene-specific analysis. Using a gene-specific primer set, transcription of each member of the ALS gene family was analyzed by real-time qPCR. Finally, the mechanism of ALS1 and ALS4 regulation was addressed by two different approaches. First, LacZ reporter strains were constructed in order to monitor more easily pH and Rim101p effects on ALS1 and ALS4 expression. Second, a tagged version of Rim101p was used to demonstrate in vivo binding of Rim101p to ALS promoters by Chromatin Immunoprecipitation (ChIP): however, no clear specific binding could be observed.