Identification des réseaux transcriptionnnels de résistance aux antifongiques chez Candida albicans

Many azole resistant Candida albicans clinical isolates overexpress genes encoding azole resistance effectors that belong to two functional categories: i) CDR1, CDR2 and MDR1, encoding azole-efflux transporters and ii) ERG11, encoding the target of azoles 14-lanosterol demethylase. The constitutive overexpression of these genes is due to activating mutations in transcription factors of the zinc cluster family (Zn2Cys6) which control their expression. Tac1p (Transcriptional activator of CDR genes 1), controlling the expression of CDR1 and CDR2, Mrr1p (Multidrug resistance regulator 1), regulating MDR1 expression and Upc2p (Uptake control 2), controlling the expression of ERG11. Another determinant of clinical azole resistance is PDR16, encoding a phospholipid transferase, whose overexpression often accompanies that of CDR1 and CDR2 in clinical isolates, suggesting that the three genes belong to the same regulon, potentially that of Tac1p. Further, MDR1 expression is not only regulated by Mrr1p, but also by the basic-leucine zipper transcription factor Cap1p (Candida activator protein 1), which controls the oxidative stress response in C. albicans and whose mutation confers azole resistance via MDR1 overexpression. These observations suggest that a complex transcriptional regulatory network controls azole resistance in C. albicans. My Ph.D. studies are aimed at identifying the direct transcriptional targets of Tac1p, Upc2p and Cap1p using genetics and functional genomics approches in order to i) characterize their regulatory network and the transcriptional modules under their direct control and ii) infer their biological functions and better understand their roles in azole resistance. In the first part of my studies, I showed that Tac1p does not only control the expression of CDR1 and CDR2, but also that of PDR16. My results also identified a new activating mutation in Tac1p (N972D) and revealed that the expression of CDR1 and PDR16 is under the control of another yet unknown regulator. The combination of transcriptomics and genome-wide location (ChIP-chip) approaches allowed me to identify the in vivo direct targets of Tac1p (PDR16, CDR1, CDR2, ERG2, others), Upc2p (ERG11, ERG2, MDR1, CDR1, others) and Cap1p (MDR1, GCY1, GLR1, others). These results also revealed that Upc2p does not only control the expression of ERG11 but also that of MDR1 and CDR1. Many new functional features of these transcription factors were found, including the constitutive binding of Tac1p to its targets under both activating and non-activating conditions, and the binding of Cap1p which extends beyond the promoter region of its target genes, to cover the open reading frame and the putative transcription termination regions, suggesting a physical interaction with the transcriptional machinery. The characterization of the transcriptional regulatory network revealed a functional interaction between these factors, notably between Cap1p and Mrr1p, and inferred potential biological functions for Tac1p (lipid mobilization and traffic, response to oxidative and osmotic stress) and confirmed or suggested other functions for Upc2p (sterol metabolism) and Cap1p (oxidative stress response, regulation of nitrogen utilization and phospholipids transport). Taken together, my results suggest that azole resistance in C. albicans is tightly linked to membrane lipid metabolism and oxidative stress response.

Keyphrases: Candida albicans, Candida albicans, antifungal agents, drug resistance, functional genomics, transcriptional regulatory networks