Phage biology and diversity in Clostridium difficile and other commensal clostridia

艰难梭菌和其他共生梭菌的噬菌体生物学和多样性

• 批准号: RGPIN-2015-06334
• 负责人: Fortier, LouisCharles
• 金额: $ 2.19万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=650980
• 关键词: Phage; biology; diversity; Clostridium; difficile

***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.

***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揭示了粪便样本中存在大量游离噬菌体颗粒，但它们的起源、宿主特异性以及对肠道微生物生态系统（微生物群）的潜在影响通常未知。同样，尽管它们的患病率很高，但它们在艰难梭菌 (Cdif) 中的作用仍然相对未被探索，而艰难梭菌是目前医疗相关性腹泻最常见的原因。我的长期目标是收集肠道细菌噬菌体和反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。我建议将我们的努力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梭菌，以及 Cdif，我们在这方面拥有强大的专业知识，并在之前的 NSERC 拨款（2007-2015 年）过程中收集了重要数据。 ***目标 1 - 我们的首要目标是从肠道细菌中分离和表征新噬菌体。我们将重点关注非致病性肠道梭菌和致病性 Cdif。我们将使用经典的 UV 和丝裂霉素 C 原噬菌体诱导策略和方案来筛选一系列人源非致病性梭菌菌株以及 Cdif 分离株。将进行宿主范围、病毒颗粒形态和基因组测序来表征这些新噬菌体，这将为我们提供有关它们在肠道中潜在作用的重要信息。***AIM 2 - 基于我们之前 NSERC 资助的 Cdif 噬菌体与宿主相互作用的工作，我们将研究 Cdif 如何响应特定前噬菌体的损失和获得。我们将使用 RNAseq 策略与 qRT-PCR、质谱和表型测定相结合来识别在溶源或生产性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 定植小鼠模型评估某些前噬菌体的获得或损失是否对肠道定植产生影响。****AIM 3 - 在我们之前的 NSERC 资助期间，我们在 Cdif 中鉴定了第一个功能性反噬菌体系统，包括保守的细胞表面蛋白 CwpV，其生物学功能仍然难以捉摸。我们将使用各种 CwpV 和噬菌体突变体并结合经典噬菌体测定（噬菌体吸附、DNA 复制、铺板效率）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 在肠道定植期间体内的影响。 ***影响。我们的研究项目将为肠道中梭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帮助我们更好地了解它们对肠道环境中 Cdif 的生活方式、毒力和竞争力的贡献。