Functional prophage and lysogen engineering in Citrobacter enabling studies of virulence and other traits

细菌引起的肠道感染很常见，也很成问题，它们可以导致轻微的致残疾病，也可以导致非常严重的——实际上是致命的——感染。大肠杆菌是一种经常登上新闻头条的致病性细菌，其中特定菌株引起各种肠道疾病，从“旅行者腹泻”、肠致病性大肠杆菌病（EPEC）到可导致肾衰竭的肠溶血性大肠杆菌病（EHEC）。这些疾病的某些方面的微生物学和分子生物学特征，但在研究进展中可能存在问题，因为没有相关感染的动物模型。这是EPEC和肠出血性大肠杆菌的一个问题，因为小鼠感染不容易或不易复制。然而，几年前，一种相关细菌（Citrobacter）的自然变异菌株在驯化的实验室小鼠中出现，该菌株（随后被归类为啮齿Citrobacter rodentium）表现出许多人类病原体的临床特征，如肠出血性大肠杆菌和肠出血性大肠杆菌，但在实验室小鼠中-一个实验可利用的系统。因此，细菌C. rodentium已经成为一些人类肠道疾病和失调的有用的小鼠替代模型，包括EPEC和EHEC，但也附带影响溃疡性结肠炎，克罗恩病，甚至结肠癌方面的研究。对啮齿c的基因组测序表明，啮齿c、EPEC和EHEC可能在一定程度上通过可移动的遗传元件（“跳跃基因”）趋同于一种共同的宿主感染策略，从而使毒力因子基因的基因组通量和运动成为可能，这为理解啮齿c作为一种模式生物及其模型大肠杆菌病原体迈出了一步。一些基因组通量被认为是由噬菌体驱动的（编码细菌病毒生产的DNA嵌入细菌染色体中）。噬菌体基因可以引起“噬菌体转化”或“溶原转化”，其中噬菌体携带的基因可以影响病原体的生理和毒力。事实上，一些噬菌体实际上是一些细菌病原体毒性的主要原因。对柠檬酸杆菌（Citrobacter）的噬菌体的分析取得了一些有限的进展，但这些噬菌体的确切性质及其对病原菌生理、毒力等性状的影响尚不清楚。这项泵启动研究将进一步表征柠檬酸杆菌的噬菌体。染色体的特定区域表明它们可能编码功能齐全的细菌病毒（噬菌体），我们最近在病原体中发现了一种新的功能齐全的噬菌体，证实了这一假设。染色体的其他区域显示了降解和不完整的病毒DNA的残余。在这个项目中，我们将制作Citrobacter的突变体，其中两个功能齐全的前噬菌体位点要么通过手术切除，要么在前噬菌体中携带特定基因的缺失（使用基因工程/编辑方法）。这些噬菌体缺失和精确突变体的银行将被表征，以确定相应突变对病原体行为的影响。这些研究将包括使用简单的实验室显微镜蠕虫进行毒力分析（在这个阶段避免使用许多实验室小鼠进行初始毒力筛选分析，这可能是不道德的）。其他生物测定将包括对啮齿鼠突变体的生理评估，包括抗生素耐药性和生物膜研究。因此，对啮齿弧菌天然噬菌体的调查和合理利用将告诉我们这些内源性病毒在驱动或调节这一重要模式肠道病原体的多种特性方面可能产生的生物学影响。此外，在未来，这项工作应该为研究通过智能工程噬菌体的溶原转化来修饰柠檬酸杆菌肠道病原体或大肠杆菌共栖物提供平台。

Gut infections due to bacteria are common and problematic and they can cause minor disabling diseases to very serious - indeed lethal - infections. E. coli is one pathogenic bacterium that regularly hits the news headlines where particular strains cause various intestinal diseases ranging from "travellers diarrhoea", and enteropathogenic E. coli disease (EPEC) to enterohaemolytic E coli disease (EHEC) that can lead to kidney failure. The microbiology and molecular biology of some aspects of these diseases are characterised but there can be problems in research progress where an animal model of the relevant infection is not available. This is an issue for EPEC and EHEC where infections of mice are not easy or reproducible. However, some years ago a natural variant strain of a related bacterium (Citrobacter) arose in domesticated laboratory mice and this strain (subsequently classified as Citrobacter rodentium) showed many of the clinical traits of the human pathogens such as EHEC and EPEC, but in the laboratory mouse - an experimentally exploitable system. Consequently, the bacterium C. rodentium has become a useful surrogate model in the mouse for some human intestinal diseases and disorders, including EPEC and EHEC but also with collateral implications for ulcerative colitis, Crohn's disease, and even research on aspects of colonic cancers. One step towards understanding C. rodentium as a model organism, and thus the E coli pathogens it models, arose with genome sequencing of C. rodentium which revealed that C. rodentium, EPEC and EHEC may have converged on a common host infection strategy partly through mobile genetic elements ("jumping genes") that enable genomic flux and movement of virulence factor genes. Some of this genomic flux was thought to be driven by prophages (where the DNA encoding production of bacterial viruses is embedded in the bacterial chromosome). Prophage genes can cause "phage conversion" or "lysogenic conversion" where genes carried by the phage can impact the physiology and virulence of the pathogen. Indeed some prophages are actually the main cause of virulence in some bacterial pathogens. Some limited progress has been made on the analysis of the prophages of Citrobacter, but the precise natures of these prophages and their impacts on the physiology, virulence and other traits of the pathogen are not clear. This pump-priming study will further characterise prophages of Citrobacter. Specific regions of the chromosome suggest that they may encode fully functional bacterial viruses (phages) and we recently confirmed this hypothesis in our discovery of a new fully functional prophage in the pathogen. Other regions of the chromosome suggest remnants of degraded and incomplete viral DNA. In this project we will make mutants of Citrobacter in which two fully functional prophage loci are either surgically removed or carry deletions in specific genes found within the prophages (using genetic engineering / editing methods). Banks of these prophage deletion and precise mutants will be characterised to define the impacts of the corresponding mutations on pathogen behaviour. These studies will include the use of simple lab-based microscopic worms for virulence assays (avoiding at this stage the, arguably unethical, use of many laboratory mice for initial virulence screening assays). Other bioassays will include physiological assessments of the C. rodentium mutants, including antibiotic resistance and biofilm studies. In this way, an interrogation and judicious exploitation of the natural prophages of C. rodentium will tell us about the possible biological impacts of these endogenous viruses in driving or modulating multiple traits of this important model enteric pathogen. Furthermore, in the future, this work should provide a platform for studies on the modification of Citrobacter gut pathogens, or E. coli commensals, through lysogenic conversion due to intelligently engineered prophages.