Phage biology and diversity in Clostridium difficile and other commensal clostridia

*背景。噬菌体(或噬菌体，即细菌病毒)对大多数细菌物种的适合性、毒性和进化都有贡献。最近的元基因组研究表明，粪便标本中存在大量的游离噬菌体颗粒，但它们的来源、宿主特异性以及对肠道微生物生态系统(微生物区系)的潜在影响通常尚不清楚。同样，尽管它们的流行率很高，但它们在艰难梭菌(CDIF)中的作用仍然相对未被探索。艰难梭菌目前是与医疗保健相关的腹泻的最常见原因。我的长期目标是收集肠道细菌噬菌体和抗噬菌体系统的功能数据，以了解它们对微生物区系动态平衡的贡献。我建议将我们的努力集中在非致病性肠杆菌，特别是那些具有免疫调节功能的肠杆菌，以及CDIF，我们在这方面拥有强大的专业知识，并在我们之前的NSERC资助过程中(2007-2015)收集了重要数据。*目标1-我们的第一个目标是从肠道细菌中分离和鉴定新的噬菌体。我们将重点关注非致病性肠杆菌，以及致病性CDIF。我们将使用经典的紫外线和丝裂霉素C原噬菌体诱导策略和方案来筛选一组来自人类的非致病性梭状芽胞杆菌菌株以及CDIF分离株。将进行宿主范围、病毒粒子形态和基因组测序来表征这些新噬菌体，这将为我们提供关于它们在肠道中潜在作用的重要信息。*目的2-在我们以前NSERC资助的CDIF中噬菌体-宿主相互作用工作的基础上，我们将研究CDIF如何对特定前驱噬菌体的丧失和获得做出反应。我们将使用RNAseq策略结合qRT-PCR、质谱学和表型分析来鉴定在溶原性或高效裂解感染期间差异表达的噬菌体和宿主蛋白。我们还将使用CDIF定植的小鼠模型来评估某些前驱体的获得或丢失是否影响肠道的定植。*目的3-在我们之前的NSERC资助期间，我们鉴定了CDIF中的第一个功能性抗噬菌体系统，包含在保守的细胞表面蛋白CwpV中，其生物学功能仍然难以捉摸。我们将使用不同的CwpV和噬菌体突变体结合经典的噬菌体分析(噬菌体吸附、DNA复制、平板效率)来表征CwpV的活性和作用机制以及与噬菌体的相互作用。我们还将评估CwpV在体内肠道定植期间的影响。*影响。我们的研究项目将为肠道中梭状芽胞杆菌噬菌体的多样性和潜在影响提供前所未有的见解。我们涉及CDIF噬菌体和抗噬菌体系统的噬菌体-宿主相互作用研究将有助于我们更好地了解它们在肠道环境中对CDIF的生活方式、毒力和竞争力的贡献。

***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.