Molecular basis of alpha5/beta1 integrin exploitation by the Helicobacter pylori type IV secretion system

幽门螺杆菌、嗜肺军团菌、猪布鲁氏菌、百日咳杆菌或亨氏巴尔通菌等细菌病原体已经进化出专门的分子机器，即IV型分泌系统(T4SS)，以定植器官并反击和破坏宿主免疫防御系统。幽门螺杆菌是一种革兰氏阴性细菌，在世界上一半的人口中定居在人的胃里。据估计，在儿童时期感染的人中，有20%会发展为消化性溃疡疾病，而胃肿瘤的发生率为2%-3%。由于这些原因，幽门螺杆菌被世界卫生组织(WHO)定义为第一类致癌物质。与最严重疾病相关的菌株含有细胞毒素相关基因cagA。CagA是由位于CAG致病岛(CagPAI)的基因编码的一种独特的蛋白质。这段CagPAI编码28种蛋白质，它们组装了一个T4SS(CagT4SS)，需要将CagA运送到宿主细胞中。转位后，CagA被宿主的Src激酶磷酸化，并上调细胞的信号系统，从而引发肿瘤的出现。因此，CagA是幽门螺杆菌感染过程中癌症发生的主要决定因素，被认为是细菌致癌的范例。CagT4SS注射CagA的机制尚不清楚。研究发现，T4SS利用β1整合素作为底物转运的宿主细胞受体。该研究计划的目的是阐明幽门螺杆菌T4SS复合体利用β1整合素的结构和分子基础。我们的研究计划建立在我们研究小组最近令人兴奋的发展基础上，并将结构研究(X射线结晶学、SAXS和EM)与相互作用研究和细胞分析联系起来。这项建议是基于三个小组目前进行的合作工作，这些工作已经导致：1)建立蛋白质纯化和相互作用研究的方案，2)对CagA结构和其β1整合素结合域的高分辨率研究，3)有效的体内检测方法来监测所研究的相互作用的功能相关性。我们的短期目标是破译(1)菌毛蛋白成分之间的相互作用的分子和结构细节，(2)它们与β1整合素受体的相互作用，以及(3)确定导致CagA注射的结合事件/激活的序列。我们的团队有着成功合作的历史，我们的专业知识具有真正的互补性，从而确保了对幽门螺杆菌整合素开发的更完整、更完整的愿景。尽管β1整合素似乎是许多病原体的共同受体，但关于这些相互作用的细节，结构信息尚不清楚。长期目标是更好地了解细菌感染的分子机制，并提供对抗这些感染迫切需要的新的治疗策略。

Bacterial pathogens such as Helicobacter pylori, Legionella pneumophila, Brucella suis, Bordetella pertusis or Bartonella henselae have evolved dedicated molecular machines, the Type IV secretion systems (T4SS), to colonise organs and counter-attack and subvert host immune defence systems. H. pylori is a Gram-negative bacterium that colonises the human stomach in half of the world population. It is estimated that 20% of individuals infected during their childhood will develop peptic ulcer disease, while gastric neoplasia will develop in 2-3%. For these reasons, H. pylori was defined as a group 1 carcinogen the by World Health Organization (WHO). Strains associated with the most severe diseases contain the cytotoxin-associated gene cagA. CagA is a unique protein encoded by a gene located in the Cag pathogenicity island (CagPAI). This CagPAI encodes for 28 proteins that assemble a T4SS (cagT4SS) required to deliver CagA into the host cell. After translocation, CagA is tyrosine phosphorylated by host Src kinases and highjacks the signalling system of the cell to provoke tumour apparition. CagA is therefore the major determinant of cancer development during H. pylori infection and is considered the paradigm of bacterial carcinogenesis. The mechanism of injection of CagA by the cagT4SS is still poorly understood. It was found that the T4SS utilises beta1 integrin as a host cell receptor for substrate translocation. The objective of the proposed research program is to elucidate the structural and molecular basis of beta1 integrin utilisation by the H. pylori T4SS complex. Our research initiative builds upon recent exciting developments in our research groups and connects structural studies (X-ray crystallography, SAXS, and EM) with interaction studies and cellular assays. The proposal is based on the current collaborative work performed by three groups that has led to: 1) established protocols for purification of the proteins and interaction studies, 2) high resolution studies of CagA structure and identification of its beta1 integrin binding domain, 3) efficient in vivo assays to monitor the functional relevance of the interactions studied.Our short-term goal is to decipher the molecular and structural details of (1) the interactions between pilus protein components (2) their interactions with the beta1 integrin receptor and (3) to determine the sequence of binding events/activation leading to CagA injection. Our groups have a history of successful collaboration and our expertises are genuinely complementary, hereby ensuring a more complete, integrated vision of the integrin exploitation by H. pylori. Although beta1 integrins appear as a common receptor for many pathogens, no structural information is available concerning the details of these interactions. The long-term objectives are to better understand the molecular mechanisms of bacterial infection and to provide new therapeutic strategies, urgently needed to combat these infections.