Phage biology and diversity in Clostridium difficile and other commensal clostridia

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***BACKGROUND. Bacteriophages (or phages, i.e. bacterial viruses), contribute to fitness, virulence, and evolution of most bacterial species. Recent metagenomic studies revealed the presence of numerous free phage particles in fecal specimens, but their origin, host specificity, and potential impact on the gut microbial ecosystem (the microbiota) are generally unknown. Likewise, their role in Clostridium difficile (Cdif), currently the most frequent cause of healthcare-associated diarrhea, has remained relatively unexplored despite their high prevalence. My long-term objective is to gather functional data on phages and antiphage systems from gut bacteria to understand their contribution to homeostasis of the microbiota. I propose to focus our efforts on non-pathogenic gut clostridia, in particular those with immunomodulatory functions, as well as Cdif, on which we have a strong expertise and collected important data in the course of our previous NSERC grants (2007-2015). ***AIM 1 - Our first aim is to isolate and characterize new phages from gut bacteria. We will focus our efforts on non-pathogenic gut clostridia, as well as pathogenic Cdif. We will use classical UV and mitomycin C prophage induction strategies and protocols to screen a collection of human-derived non-pathogenic clostridial strains as well as Cdif isolates. Host range, virion morphology, and genome sequencing will be done to characterize these new phages, which will give us important information about their potential role in the gut.***AIM 2 - Building upon our previous NSERC-funded work on phage-host interactions in Cdif, we will study how Cdif responds to the loss and gain of specific prophages. We will use an RNAseq strategy combined with qRT-PCR, mass spectrometry and phenotypic assays to identify phage and host proteins that are differentially expressed during lysogeny or during a productive lytic infection. We will also assess whether the gain or loss of certain prophages impacts on colonization of the gut using a mouse model of Cdif colonization.****AIM 3 - During our previous NSERC grant, we identified the first functional antiphage system in Cdif, consisting in the conserved cell surface protein CwpV, for which a biological function remains elusive. We will characterize the activity and mechanism of action of CwpV as well as the interaction with phages using various CwpV and phage mutants combined with classical phage assays (phage adsorption, DNA replication, efficiency of plating). We will also assess the impact of CwpV in vivo during colonization of the gut. ***IMPACT. Our research project will provide unprecedented insight into the diversity and potential impact of clostridial phages in the gut. Our phage-host interactions studies involving Cdif phages and antiphage systems will help us better understand their contribution in the lifestyle, virulence and competitiveness of Cdif within the gut environment.