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。细菌病毒或噬菌体是生物圈中最丰富的生物实体。它们给与之共存的敏感细菌带来了巨大的压力。严格裂解的噬菌体可以消灭整个细菌种群，从而破坏海洋等生态系统中微生物的平衡。温带噬菌体可以采取溶源生活方式，在此期间它们作为“休眠”原噬菌体隐藏在宿主染色体内。许多原噬菌体在溶源过程中改变其宿主的生理学和/或毒力，这种现象在某些病原体中已有充分记录。值得注意的是，原噬菌体重新激活（诱导）可以随时发生，导致裂解周期的启动，导致宿主死亡。据报道，肠道中噬菌体密度较高，有时与某些慢性疾病（例如克罗恩氏结肠炎）有关。由于大多数肠道细菌是溶原性的，原噬菌体诱导可能解释了这些观察结果，但影响这种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原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。方法论。我将研究特定前噬菌体对两种致病性梭状芽胞杆菌的生理和适应性的影响：产气荚膜梭菌（目标 1）和艰难梭菌（Cd）（目标 2）。我还将探索与肠道生理学相关的非致病性共生梭状芽胞杆菌中的原噬菌体（目标 3）。将首先在细菌基因组中通过计算机识别原噬菌体，然后诱导和分离病毒颗粒。这些噬菌体将被重新引入易感菌株中以产生新的溶原菌。然后将评估各种表型，例如生长、孢子形成、孢子萌发、生物膜形成、移动性和毒素产生。将鉴定参与细菌表型调节的原噬菌体基因，并进一步表征其产物。对于共生梭状芽胞杆菌，将通过分析公开可用的相应噬菌体存在的病毒组数据集，以计算机方式推断体内原噬菌体诱导。最后，我将破译 Cd 噬菌体与其宿主受体、表面层蛋白 SlpA 之间的分子相互作用（目标 4）。我将使用一个独特的 Cd 模型，其中 slpA 基因已发生突变。将进行各种 slpA 基因构建的互补，然后进行感染和吸附测定。还将进行噬菌体颗粒的冷冻电子显微镜检查和受体结合蛋白的 X 射线晶体学检查。 影响。更好地表征原噬菌体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。对噬菌体与宿主相互作用的深入研究对于未来开发基于噬菌体的基因工程工具非常有价值，例如，可以消除特定细菌种类以评估其在肠道中的生理作用。