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，也很有问题，它们可以导致轻微的致残性疾病，甚至是非常严重的--致命的--感染。大肠杆菌是一种经常登上新闻头条的致病细菌，特定的菌株会导致各种肠道疾病，从“旅行者腹泻”、肠源性大肠杆菌病(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是有特点的，但在研究进展中可能会出现问题，因为没有相关感染的动物模型。对于EPEC和EHEC来说，这是一个问题，因为感染小鼠不容易或不容易复制。然而，几年前，一种相关细菌的自然变异菌株(柠檬酸杆菌)在驯化的实验室小鼠中出现，该菌株(后来被归类为轮状柠檬酸柠檬酸杆菌)表现出许多人类病原体的临床特征，如EHEC和EPEC，但在实验室小鼠中--一个可实验利用的系统。因此，罗氏杆菌已经成为一些人类肠道疾病和障碍的有用的小鼠替代模型，包括EPEC和EHEC，但也对溃疡性结肠炎、克罗恩病，甚至结肠癌方面的研究有间接影响。了解轮状芽胞杆菌作为模式生物的一步，以及它所模拟的大肠杆菌病原体，出现在轮状芽胞杆菌的基因组测序中，这揭示了轮状芽胞杆菌、EPEC和EHEC可能部分通过使基因组流动和毒力因子基因移动的可移动遗传元件(“跳跃基因”)在共同的宿主感染策略上趋同。其中一些基因组流量被认为是由前驱体(编码产生细菌病毒的DNA嵌入在细菌染色体中)驱动的。噬菌体基因可引起“噬菌体转换”或“溶源转换”，即噬菌体携带的基因可影响病原体的生理和毒力。事实上，一些前驱体实际上是一些细菌病原体致病的主要原因。在柠檬酸杆菌前驱体的分析方面取得了一些有限的进展，但这些前驱体的确切性质及其对病原体的生理、毒力和其他特征的影响尚不清楚。这项泵启动研究将进一步描述柠檬酸杆菌的前驱噬菌体。染色体的特定区域表明它们可能编码全功能细菌病毒(噬菌体)，我们最近在病原体中发现了一个新的全功能噬菌体，证实了这一假设。染色体的其他区域表明残留的降解和不完整的病毒DNA。在这个项目中，我们将制作柠檬酸杆菌的突变体，其中两个完全功能的前噬菌体基因座要么通过手术移除，要么携带在前驱体内发现的特定基因的缺失(使用基因工程/编辑方法)。将对这些前噬菌体缺失和精确突变的库进行表征，以定义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微型蠕虫进行毒力分析(在这个阶段避免使用许多实验室小鼠进行最初的毒力筛选测试，这是不道德的)。其他生物检测将包括对轮状芽胞杆菌突变株的生理评估，包括抗生素耐药性和生物被膜研究。通过这种方式，对轮状芽胞杆菌天然前驱体的询问和明智的利用将告诉我们这些内源性病毒在驱动或调节这种重要的模式肠道病原体的多种特征方面可能产生的生物学影响。此外，在未来，这项工作将为利用智能工程前驱体通过溶原性转化来修饰柠檬酸杆菌肠道病原体或大肠杆菌共生体的研究提供一个平台。

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.