Phage-host interactions in pathogenic and commensal clostridia

背景细菌病毒，或称细菌，是生物圈中最丰富的生物实体。它们对与它们共存的易感细菌施加了巨大的压力。严格意义上的溶解性细菌可以大量消灭整个细菌种群，从而破坏海洋等生态系统中的微生物平衡。温带嗜热菌可以采取溶原性的生活方式，在此期间，它们作为“休眠”的前噬菌体隐藏在宿主染色体内。许多原噬菌体在溶原性过程中改变其宿主的生理学和/或毒力，这是在某些病原体中有充分记载的现象。值得注意的是，原噬菌体再活化（诱导）可以在任何时间发生，导致裂解周期的启动，导致其宿主死亡。据报道，肠道中存在高噬菌体密度，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的前噬菌体对两种致病性梭菌的生理和适应性的影响：产气荚膜梭菌（Aim 1）和艰难梭菌（Cd）（Aim 2）。我还将探索在收集的非致病性肠道梭菌的肠道生理相关的前噬菌体（目的3）。将首先在细菌基因组中通过计算机识别proximal，然后诱导和分离病毒颗粒。这些细菌将被重新引入敏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。参与细菌表型调节的前噬菌体基因将被鉴定，并对其产物进行进一步表征。对于嗜热梭菌，将通过分析可公开获得的病毒组数据集以确定相应的噬菌体的存在，在计算机模拟中推断体内原噬菌体诱导。最后，我将破译分子之间的相互作用镉及其宿主受体，表面层蛋白SlpA（Aim 4）。我将使用一个独特的镉模型，其中slpA基因已经突变。将进行与各种slpA基因构建体的互补，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和受体结合蛋白的X射线晶体学。 冲击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群稳定性的影响。噬菌体-宿主相互作用的深入研究将对未来开发基于基因工程的噬菌体工具非常有价值，例如，耗尽特定细菌物种以评估其在肠道中的生理作用。

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.