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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740818
• 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.

背景资料。细菌病毒或噬菌体是生物圈中最丰富的生物实体。它们给与它们共生的敏感细菌带来了巨大的压力。严格的裂解噬菌体可以杀死整个细菌种群，从而破坏海洋等生态系统中微生物平衡的稳定。温带噬菌体可以采取溶源的生活方式，在此期间，它们作为“休眠”的先知隐藏在它们的宿主染色体中。许多前驱体在溶原性过程中改变其宿主的生理和/或毒力，这一现象在某些病原体中得到了很好的记录。值得注意的是，原噬菌体的重新激活(诱导)可以在任何时候发生，导致裂解循环的开始，导致宿主死亡。据报道，肠道中有高密度的噬菌体，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，原噬菌体诱导可能解释了这些观察到的现象，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原噬菌体的多样性和活性，并评估它们对微生物区系组成的影响。方法论。我将研究特定的前驱体对两种致病性梭状芽孢杆菌的生理学和适合性的影响：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集合中的预言体(目标3)。将首先在细菌基因组中鉴定前驱体，然后诱导和分离病毒颗粒。这些噬菌体将被重新引入敏感菌株，以产生新的溶原物。然后将评估各种表型，如生长、产孢量、孢子萌发、生物膜形成、迁移率和毒素产生。参与调节细菌表型的原噬菌体基因将被识别，其产物将被进一步表征。对于共生性梭状芽孢杆菌，通过分析可公开获得的相应噬菌体存在的病毒体数据集，将在电子计算机中推断体内原噬菌体的诱导。最后，我将破译CD噬菌体和它们的宿主受体--表层蛋白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，在该模型中SLPA基因发生了突变。随后将与各种SLPA基因构建互补，然后进行感染和吸附分析。还将进行噬菌体颗粒的冷冻电子显微镜和受体结合蛋白的X射线结晶学研究。冲击力。更好地表征原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区系稳定性的影响。对噬菌体-宿主相互作用的深入研究将对未来基于基因工程的噬菌体工具的开发具有非常重要的价值，例如，能够耗尽特定细菌物种来评估它们在肠道中的生理作用。