Phage biology and diversity in Clostridium difficile and other commensal clostridia

* * *的背景。噬菌体（或噬菌体，即细菌病毒）有助于大多数细菌物种的适应性、毒力和进化。最近的宏基因组研究表明，粪便标本中存在大量游离噬菌体颗粒，但它们的来源、宿主特异性以及对肠道微生物生态系统（微生物群）的潜在影响通常是未知的。同样，它们在艰难梭菌（Cdif）中的作用，目前是医疗保健相关腹泻的最常见原因，尽管它们的患病率很高，但仍然相对未被探索。我的长期目标是收集肠道细菌中噬菌体和抗噬菌体系统的功能数据，以了解它们对微生物群稳态的贡献。我建议将我们的工作重点放在非致病性肠道梭菌上，特别是那些具有免疫调节功能的肠道梭菌，以及Cdif，我们在之前的NSERC资助（2007-2015）中拥有强大的专业知识和收集的重要数据。***目标1 -我们的第一个目标是从肠道细菌中分离和表征新的噬菌体。我们将把重点放在非致病性肠道梭菌和致病性Cdif上。我们将使用经典的紫外线和丝裂霉素C前噬菌体诱导策略和方案筛选人类来源的非致病性梭菌菌株和Cdif分离株。宿主范围、病毒粒子形态和基因组测序将对这些新的噬菌体进行表征，这将为我们提供有关它们在肠道中潜在作用的重要信息。***目标2 -基于我们之前nserc资助的关于Cdif中噬菌体-宿主相互作用的工作，我们将研究Cdif如何响应特定噬菌体的损失和获得。我们将使用RNAseq策略结合qRT-PCR，质谱和表型分析来鉴定在溶原过程中或在高效溶菌感染过程中差异表达的噬菌体和宿主蛋白。我们还将利用小鼠Cdif定植模型来评估某些噬菌体的增加或减少是否会影响肠道的定植。****AIM 3 -在我们之前的NSERC资助期间，我们在Cdif中发现了第一个功能性的噬菌体系统，由保守的细胞表面蛋白CwpV组成，其生物学功能仍然难以捉摸。我们将利用各种CwpV和噬菌体突变体，结合经典的噬菌体检测（噬菌体吸附、DNA复制、电镀效率），表征CwpV的活性、作用机制以及与噬菌体的相互作用。我们还将评估CwpV在肠道定植过程中的体内影响。* * *的影响。我们的研究项目将为肠道中梭状噬菌体的多样性和潜在影响提供前所未有的见解。我们的噬菌体-宿主相互作用研究涉及Cdif噬菌体和抗噬菌体系统，将有助于我们更好地了解它们在肠道环境中Cdif的生活方式、毒力和竞争力方面的贡献。

***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.