Phage-host interactions in pathogenic and commensal clostridia

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Background. Viruses of bacteria, or phages, are the most abundant biological entities of the biosphere. They put a huge pressure on susceptible bacteria cohabiting with them. Strictly lytic phages can decimate whole bacterial populations, thereby destabilizing the microbial equilibrium in ecosystems such as the oceans. Temperate phages can adopt a lysogenic lifestyle, during which they hide as "dormant" prophages within their host chromosome. Many prophages modify the physiology and/or virulence of their host during lysogeny, a phenomenon well documented in certain pathogens. Of note, prophage reactivation (induction) can occur at any time, resulting in the initiation of a lytic cycle causing death of their host. High phage densities have been reported in the gut, sometimes associated with certain chronic diseases (e.g. Crohn's colitis). Since most gut bacteria are lysogenic, prophage induction likely explains these observations, but the factors influencing this phenomenon are unknown. My long-term goal is to characterize the diversity and activity of prophages infecting gut bacteria and assess their impact on the composition of the microbiota. Methodology. I will study the impact of specific prophages on the physiology and fitness of two pathogenic clostridia: Clostridium perfringens (Aim 1) and Clostridioides difficile (Cd) (Aim 2). I will also explore prophages in a collection of non-pathogenic commensal clostridia of relevance to the gut physiology (Aim 3). Prophages will be first identified in silico in bacterial genomes, followed by induction and isolation of viral particles. These phages will be re-introduced into susceptible strains to create new lysogens. Various phenotypes will then be assessed, such as growth, sporulation, spore germination, biofilm formation, mobility and toxin production. The prophage genes involved in the modulation of bacterial phenotypes will be identified and their products further characterized. For commensal clostridia, in vivo prophage induction will be inferred in silico by analyzing virome datasets publicly available for the presence of the corresponding phages. Finally, I will decipher the molecular interaction between Cd phages and their host receptor, the surface layer protein SlpA (Aim 4). I will use a unique Cd model in which the slpA gene has been mutated. Complementation with various slpA gene constructions followed by infection and adsorption assays will be done. Cryo-electron microscopy of phage particles and X-ray crystallography of receptor-binding proteins will also be performed. Impact. A better characterization of prophage diversity and activity will help understand their impact on gut bacteria and on stability of the microbiota. An in-depth study of phage-host interactions will be highly valuable for the future development of genetically engineered phage-based tools enabling, for example, depletion of specific bacterial species to assess their physiological role in the gut.