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

细菌病原体如幽门螺杆菌、嗜肺军团菌、猪布鲁氏菌、百日咳博德特氏菌或汉赛巴尔通体已经进化出专用的分子机器，IV型分泌系统（T4 SS），以定殖器官并反击和破坏宿主免疫防御系统。H.幽门螺杆菌是一种革兰氏阴性细菌，在世界一半人口的胃中定植。据估计，20%在儿童时期感染的个体将发展为消化性溃疡疾病，而胃肿瘤将在2- 3%中发展。由于这些原因，H。幽门螺杆菌被世界卫生组织（WHO）定义为1组致癌物。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）中的基因编码的独特蛋白。这种CagPAI编码28种蛋白质，其组装将CagA递送到宿主细胞中所需的T4 SS（cagT 4SS）。易位后，CagA被宿主Src激酶酪氨酸磷酸化，并劫持细胞的信号传导系统，引起肿瘤出现。因此，CagA是H.幽门螺杆菌感染，被认为是细菌致癌的范例。CagA通过cagT 4SS注射的机制仍然知之甚少。发现T4 SS利用β 1整联蛋白作为底物易位的宿主细胞受体。该研究计划的目的是阐明β 1整合素的结构和分子基础的利用H。pylori T4 SS复合体。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射线晶体学，SAXS和EM）与相互作用研究和细胞分析联系起来。该提案基于三个小组目前开展的协作工作，这些工作已导致：1）建立了蛋白质纯化和相互作用研究的方案，2）CagA结构的高分辨率研究和其β 1整联蛋白结合结构域的鉴定，3）有效的体内测定来监测所研究的相互作用的功能相关性。术语目标是破译（1）菌毛蛋白组分之间的相互作用（2）它们与β 1整联蛋白受体的相互作用和（3）确定导致CagA注射的结合事件/激活的序列的分子和结构细节。我们的团队有着成功合作的历史，我们的专业知识是真正的互补，从而确保H.幽门。虽然β 1整合素作为许多病原体的共同受体出现，但没有关于这些相互作用细节的结构信息。长期目标是更好地了解细菌感染的分子机制，并提供新的治疗策略，迫切需要打击这些感染。

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.