This project is built upon our recent genome-wide identification of a great number (more than 200) and a large diversity of potential regulatory RNAs in C. difficile. By combining in silico analysis, RNAseq and genome-wide promoter mapping we have identified ncRNAs in intergenic regions, cis-antisense RNAs, riboswitches (5’-cis-regulatory elements) and trans riboregulators requiring the RNA chaperone protein Hfq (Soutourina et al, PLoS Genetics, 2013 ; Soutourina, Current Opinion Microbiol, 2017 ; Soutourina et al, Frontiers in Microbiology, 2020). These ncRNAs might play important roles in the control of gene expression during the C. difficile infection cycle including metabolic adaptations, biofilm formation, stress responses, defence mechanisms and sporulation. Our goal is to determine the biological roles of regulatory RNAs and to uncover the molecular mechanisms of RNA-based regulations employed by C. difficile outside and inside the host. We are particularly interested in original aspects of RNA-based control in C. difficile and will focus on i) the function and regulation of C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system for defence against foreign DNA and type I toxin-antitoxin systems; ii) the role of the c-di-GMP signalling pathway mediated by ncRNAs in cellular processes associated with community behaviour; iii) the RNA chaperone protein Hfq-dependent ncRNA network controlling key steps of the C. difficile infection cycle and the role of ncRNAs specific to the epidemic strain 027. We propose an integrative approach to address the mechanisms controlling the C. difficile pathogenesis at the molecular, cellular and community levels (Soutourina, Current Opinion Microbiol, 2017). After the identification of the molecular players in this RNA-based control: regulatory RNAs, the RNA chaperone protein Hfq and specific ribonucleases, we will look at the dynamics of expression of these regulatory elements during C. difficile infection cycle and their role for the C. difficile interactions with other components of gut communities including numerous bacteriophages.