Phage-host interactions in pathogenic and commensal clostridia

致病性和共生梭菌中噬菌体与宿主的相互作用

• 批准号: RGPIN-2020-05776
• 负责人: Fortier, LouisCharles
• 金额: $ 2.62万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760925
• 关键词: Phage; host; interactions; pathogenic; commensal

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.

背景。细菌的病毒或噬菌体是生物圈中最丰富的生物实体。它们对与它们共生的易感细菌施加了巨大的压力。严格的裂解噬菌体可以摧毁整个细菌种群，从而破坏生态系统（如海洋）中的微生物平衡。温带噬菌体可以采用溶原性的生活方式，在此期间，它们作为“休眠”的噬菌体隐藏在宿主染色体内。许多噬菌体在溶原过程中改变宿主的生理和/或毒力，这一现象在某些病原体中有充分的记载。值得注意的是，噬菌体的再激活（诱导）可以在任何时候发生，导致分解周期的启动，导致宿主死亡。据报道，在肠道中存在高噬菌体密度，有时与某些慢性疾病（如克罗恩结肠炎）有关。由于大多数肠道细菌都是溶原性的，噬菌体诱导可能解释了这些观察结果，但影响这一现象的因素尚不清楚。我的长期目标是表征感染肠道细菌的前噬菌体的多样性和活性，并评估它们对微生物群组成的影响。方法。我将研究特异性噬菌体对两种致病性梭状芽孢杆菌：产气荚膜梭状芽孢杆菌（Clostridium perfringens, Aim 1）和艰难梭状芽孢杆菌（clostridiides difficile, Cd）的生理和适应性的影响。我还将探讨与肠道生理学相关的非致病性共生梭菌集合中的噬菌体（目的3）。噬菌体将首先在细菌基因组中进行计算机鉴定，然后进行病毒颗粒的诱导和分离。这些噬菌体将被重新引入易感菌株以产生新的溶原。然后将评估各种表型，如生长、产孢、孢子萌发、生物膜形成、流动性和毒素产生。参与细菌表型调节的前噬菌体基因将被鉴定，其产物将进一步表征。对于共生梭菌，体内噬菌体诱导将通过分析相应噬菌体存在的公开病毒体数据集来推断。最后，我将破译Cd噬菌体与其宿主受体（表层蛋白SlpA）之间的分子相互作用（Aim 4）。我将使用一种独特的Cd模型，其中slpA基因发生了突变。将与各种slpA基因结构进行互补，然后进行感染和吸附试验。噬菌体颗粒的冷冻电子显微镜和受体结合蛋白的x射线晶体学也将进行。的影响。更好地表征前噬菌体的多样性和活性将有助于了解它们对肠道细菌和微生物群稳定性的影响。对噬菌体-宿主相互作用的深入研究将对未来基于噬菌体的基因工程工具的发展具有很高的价值，例如，利用特定细菌物种的消耗来评估它们在肠道中的生理作用。