"Molecular characterization of a novel host cell binding and invasion factor by the major foodborne pathogen Campylobacter jejuni"

空肠弯曲杆菌引起的食源性感染是人类细菌性胃肠炎的主要原因，每年可能导致全球多达4亿至5亿例病例。疾病的结果从轻度、非炎症性、自限性腹泻到重度、炎症性、血性腹泻和持续数周的腹痛不等，但C。空肠弯曲还与更严重的后遗症相关，例如在某些个体中发生反应性关节炎和周围神经病、Miller-Fisher和Guillain-Barrè综合征。主要的挑战是缺乏有效的C。空肠在食物链中的作用，以及对宿主-病原体相互作用的基本机制的理解相对较差。尽管过去报道了一系列细菌毒力因子，但它们在发病机制中的潜在作用在文献中经常是有争议的，并且仍然不完全清楚。例如，它仍然是广泛未知的建议C。空肠因子是否为典型的粘附素，以及这些因子调控哪些疾病相关的信号通路。最近，我们鉴定了一种新的C。空肠毒力因子HtrA是一种与宿主细胞直接相互作用的表面暴露丝氨酸蛋白酶。本研究在建立了一系列重要的分子工具和方法的基础上，对HtrA在C.离体和体内空肠。特别是，我们已经确定了HtrA中的精氨酸-甘氨酸-天冬氨酸（RGD）基序，我们的初步数据表明，该基序是表面暴露的，对细胞结合和C的入侵至关重要。空肠。因此，我们建议HtrA和它的RGD基序作为识别位点的宿主细胞受体整合素-b1。我们生成了C。空肠菌株表达HtrA变体，在RGD基序或蛋白酶失活的S197 A中具有点突变，将使用体外结合和细胞侵袭测定、定量免疫沉淀结合敲除（QUICK）、信号传导方法、共聚焦显微镜和活细胞成像以及短期和长期小鼠感染实验对其进行进一步分析。这种方法将使我们能够重建发生在病原体-宿主细胞界面的事件序列，并确定新的关键致病性决定因素。进一步了解细菌毒力因子的生物学意义和细菌与宿主相互作用的性质，将导致新的检测，控制和减少弯曲杆菌感染的策略。从长远来看，这可能有助于减少弯曲杆菌引起的人类疾病的负担，并减少临床费用。

Food-borne infections by Campylobacter jejuni are the major cause of human bacterial gastroenteritis, and may be responsible for as many as 400-500 million cases worldwide every year. Disease outcomes vary from mild, non-inflammatory, self-limiting diarrhea to severe, inflammatory, bloody diarrhea and abdominal pain lasting for several weeks, but C. jejuni is also associated with more severe sequelae such as the development of reactive arthritis and peripheral neuropathies, the Miller-Fisher and Guillain-Barrè syndromes, in some individuals. Major challenges are the lack of effective control schemes for C. jejuni in the food chain and a relatively poor understanding of basic mechanisms underlying host-pathogen interactions. Despite the array of bacterial virulence factors reported in the past, their potential role in pathogenesis is often controverse in the literature and still not fully clear. For example, it remained widely unknown which of the proposed C. jejuni factors are typical adhesins or not, and which disease-related signaling pathways are regulated by these factors. Recently, we have identified a novel C. jejuni virulence factor directly interacting with host cells, high temperature requirement A (HtrA), a surface exposed serine protease. Having established a series of important molecular tools and methods in our previous studies, this proposal is designed to gain a detailed understanding of the molecular mechanisms underlying specific HtrA functions in cell adhesion, invasion and host signaling during infection with C. jejuni in vitro and in vivo. In particular, we have identified an arginine-glycine-aspartate (RGD) motif in HtrA, and our preliminary data indicate that this motif is surface-exposed and crucial for cell binding and invasion by C. jejuni. We therefore propose that HtrA and its RGD-motif serve as recognition site for the host cell receptor integrin-b1. We generated C. jejuni strains expressing HtrA variants with point mutations in the RGD-motif or protease-inactive S197A, which will be further analysed using in vitro binding and cell invasion assays, quantitative immunoprecipitation combined with knockdown (QUICK), signaling methods, confocal microscopy and live cell imaging as well as short- and longterm mouse infections experiments. This approach will allow us to reconstruct the sequence of events occurring at the pathogen-host cell interface, and to identify novel key pathogenicity determinants. Further progress both in understanding of the biological significance of bacterial virulence factors and the nature of bacterial interactions with its host will lead to new strategies for detecting, controlling, and reducing Campylobacter infections. In a longterm perspective, this could help both to decrease the burden of Campylobacter-induced human illness and to reduce clinical expenses.