EspO orthologs and NleF: type III secretion system effectors of enteric pathogens that modulate apoptosis and inflammation

大肠杆菌是一种经常存在于温血动物肠道中的细菌。大肠杆菌的亚群已经进化出了致病的能力。肠出血性大肠杆菌(EHEC)就是其中之一，它可以引起人类的血性腹泻。感染可能涉及影响肾脏的危及生命的并发症，并经常通过食物链感染。肠源性大肠杆菌(EPEC)菌株是导致发展中国家婴儿急性水样腹泻的一个相关细菌亚群。志贺氏菌与大肠杆菌关系密切，会引起细菌性痢疾(又名志贺氏菌病)。志贺氏菌病通常是通过摄入受污染的食物或水感染的，它仍然是一种全球地方病，每年导致约1.65亿例严重痢疾和100万人死亡，特别是在五岁以下儿童中。非伤寒沙门氏菌(NTS)菌株每年估计导致10亿例自限性食源性胃肠炎。最近，多重耐药菌株已成为致命性侵袭性菌血症的重要原因，特别是在撒哈拉以南地区。重要的是，虽然被认为是高度优先的，但目前还没有针对这两种病原体的疫苗，这些病原体的发展依赖于更好地了解它们的生物学和感染策略。EPEC、EHEC、志贺氏菌和沙门氏菌依靠“分子注射器”在肠道定植并产生疾病。这种注射器用于将一组称为效应器的细菌蛋白质注射到肠道内的细胞中。这一过程被称为III型分泌(T3S)，使细菌能够为了自己的利益控制宿主细胞内的过程。我们的研究表明，T3S对于EHEC和EPEC在肠道衬里的黏附和干扰宿主反应的诱导至关重要，否则宿主反应可能会解决感染。受感染的宿主通过增强免疫反应来对抗细菌感染，而受感染的细胞可能通过程序性细胞死亡(又名细胞凋亡)来帮助清除病原体。重要的是，低水平的炎症可能有助于病原体在宿主体内建立立足点，因为它调节肠道生理和正常的肠道菌群。因此，致病的大肠杆菌、志贺氏菌和沙门氏菌注射T3S效应蛋白也就不足为奇了，T3S效应蛋白既能破坏炎症，也能破坏细胞凋亡。我们最近发现EPEC和EHEC T3SS效应器NleF与宿主细胞蛋白caspase-4结合，caspase-4在细胞凋亡和炎症中发挥作用。同样，我们发现EHEC的效应蛋白Espo，其家族成员志贺氏菌(OSPE)和沙门氏菌(SOPO)与宿主细胞蛋白HAX-1结合，HAX-1是一种主要的凋亡抑制因子，在宿主免疫反应中起间接作用。这项工作的广泛目的是描述作用机制，并了解Espo家庭成员和NleF在感染过程中的作用。特别是，我们的目标(这也是我们的强项)是将体外生化和细胞生物学检测获得的结果转化为体内病原体与宿主的相互作用。具体目标包括：1.确定Espo：HAX-1和NleF：caspase-4复合体的结构；2.确定Espo家族成员和NleF3的细胞内相互作用组。使用细胞培养模型剖析Espo家族成员和NleF细胞信号通路4。Espo、SOPO和NleF及其宿主细胞伙伴蛋白在动物体内病原-宿主相互作用中的作用研究

Escherichia coli is a bacterium that often inhabits the intestines of warm-blooded animals. Subsets of E. coli have evolved the ability to cause disease. One such group are enterohaemorrhagic E. coli (EHEC), which can cause bloody diarrhoea in humans. Infections can involve life-threatening complications affecting the kidneys and are frequently acquired via the food chain. Enteropathogenic E. coli (EPEC) strains are a related subset of bacteria that cause acute watery diarrhoea in infants in the developing world. Shigella strains, which are closely related to E. coli, cause bacillary dysentery (aka Shigellosis). Shigellosis, which is usually acquired by ingestion of contaminated food or water, remains a global endemic disease responsible for ca. 165 million cases of severe dysentery and 1 million deaths annually, particularly in children less than five years of age. Non-typhoidal Salmonella enterica (NTS) strains cause an estimated 1 billion cases of self-limiting foodborne gastroenteritis annually. More recently, multiple antibiotic-resistant strains have emerged as important causes of fatal invasive bacteraemia, particularly in sub-Saharan regions. Importantly, although considered high priority, no vaccines are yet available for either of these pathogens, the development of which relies on better understanding of their biology and infection strategies.EPEC, EHEC, Shigella and Salmonella rely on a 'molecular syringe' to colonise the intestines and produce disease. This syringe serves to inject a set of bacterial proteins termed effectors into cells lining the intestines. This process, known as Type III secretion (T3S), enables the bacteria to take control of processes inside host cells for their own benefit. Our research has shown that T3S is vital for adherence of EHEC and EPEC to the gut lining and to interfere with the induction of host responses that might otherwise resolve the infection. Infected hosts fight bacterial infection by mounting immune responses while infected cells might assist in clearing the pathogen by undergoing programme cell death (aka apoptosis). Importantly, low-level inflammation may assist the pathogens in establishing a foothold within the host as it modulates the gut physiology and the normal gut flora. It is therefore not surprising that the pathogenic E. coli, Shigella and Salmonella inject T3S effector proteins that subvert both inflammation and apoptosis. We recently found that the EPEC and EHEC T3SS effector NleF binds a host cell protein called caspase-4, which plays a role in both apoptosis and inflammation. Similarly, we found the effector EspO of EHEC, and it's family members in Shigella (OspE) and Salmonella (SopO) bind a host cell protein called Hax-1, which is a major inhibitor of apoptosis and has an indirect role in host immune responses. The broad aim of this work is to characterise the mechanism of action and to understand the role during infection of the EspO family members and NleF. In particular, our aim (which is also our strength) is to translate results obtained by biochemical and cell biology assays in vitro to pathogen-host interactions in vivo. The specific aims include: 1. Determine the structure of the EspO:Hax-1 and NleF:caspase-4 complexes 2. Determine the intracellular interactome of EspO family members and NleF3. Dissect the EspO family members and NleF cell signalling pathways using cell culture models4. Study the role of EspO, SopO and NleF and their host cell partner proteins in pathogen-host interaction in animal models in vivo