Clostridioides difficile (CD) is the major cause of nosocomial infections associated with antibiotic therapy. The disruption of the colonic microbiota by antibiotics promotes colonization of the gut by CD. Many aspects of CD pathogenesis remain poorly understood. During the infection CD survives in phage-rich gut communities by relying on defense systems like CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) for adaptive prokaryotic immunity. Toxin-Antitoxin (TA) and abortive infection (Abi) systems also contribute to prophage maintenance, prevention of phage infection, and stress response. RNAs have emerged as key components of these defense systems. CRISPR RNAs in complex with Cas proteins interfere with phage infection by targeting foreign nucleic acid for destruction. In type I TA (TITA), the antitoxin is a small antisense RNA that neutralizes toxin mRNA by inhibiting its translation and promoting its degradation and in type III TA, antitoxin RNA form a complex with the toxin leading to protein sequestration. Prokaryotic defense systems cluster together forming defense islands and could be functionally linked. This project is built upon our preliminary data on CRISPR-Cas, TITA and Abi-like systems in CD frequently associated with prophages. Our goal is to decipher the interplay between these systems and their contribution to CD adaptation and interactions with phages. We will use an integrative strategy combining genomics, molecular biology, genetics, bioinformatics and animal models. We expect to identify the roles of RNA-based defense systems with associated protein machineries in CD contributing to its fitness inside the host and enlarge the study to cover evolutionary aspects of these mechanisms. These data will shed new light on the coordination of bacterial defense strategies and genome evolution, pertinent for the development of phage and genome editing, epidemiological monitoring and new therapeutic strategies.