SSA Investigating the role of the bacterial mechanosensitive channel Ynal in Salmonella pathogenesis.

在这个项目中，我们将第一次研究机械敏感通道在微生物发病机制中的作用。肠沙门氏菌(沙门氏菌)是一种食源性病原体，每年与人类约2700万例伤寒和近1亿例胃肠炎有关。大多数肠道沙门氏菌血清型可以感染广泛的脊椎动物宿主，包括作为主要感染源的食物生产动物。在农场动物中，一些血清型会引起类似伤寒的全身性感染，损害福利和生产力。然而，这些细菌能够在宿主上定居并引发疾病的分子机制还需要进一步研究。机械敏感(MSC)通道在整个生命王国中无处不在。它们被膜张力的变化所控制，而在高等生物中，它们参与了听力、平衡和痛觉等过程。在细菌细胞中，它们需要在低渗透冲击下生存，例如从高盐环境转移到低盐环境。在这种情况下，除非MS通道打开释放细胞溶质，否则细菌细胞就会溶解并死亡。MSCL和MSCs通道是参与这一反应的主要通道。在大肠杆菌中，它们在生化、结构和遗传水平上得到了广泛的研究。细菌有多个MSCs家族成员，如大肠杆菌和沙门氏菌有6个成员，包括YNAI(STM1663)。这些家族通过它们共同的核心域结构相互联系，但通过额外的结构域区分开来，这些结构域通常功能未知。这种结构的多样性以及与之相关的功能变化的潜力还没有被很好地理解。最近的两项研究表明，在养殖动物细菌感染过程中，其中一个通道YNAI是宿主组织定植和/或致病所必需的。Chaudhuri等人对鼠伤寒沙门氏菌进行了转座子定向插入位点测序(TRADIS)，并确定了转座子引起减毒的基因2。在猪、牛和鸡的肠道定植中，多次独立插入会损害肠道定植。在巨噬细胞(http://tinyurl.com/HintonLabSalCom).)中，ynaI的转录水平略有增加在另一项研究中，空肠弯曲杆菌中的YNAI同源物被发现是鸡定植所必需的。与MSCs通道相比，人们对YNAI的了解较少，但表现出独特的电和生理特性。YNAI通道具有低电导(~2pA，MSCs=25pA)，并且在切除的贴片中需要高压力来栅极，接近于否则将溶解细胞的压力。该蛋白由5个跨膜区组成，其中TM3-5与MSCs关系密切。TM5是毛孔衬里序列，连接到广泛的C-末端细胞质结构域。与MSCs相关蛋白的典型通道一样，该通道是一种同源七聚体。冷冻电子显微镜研究显示，YNAI的胞液结构域与MSCs的结构相似，提示每个亚基的TM1-2与其余3个TMS倾斜。对于MSCs，已经提出了类似的门控机制。本项目旨在了解YNAI在沙门氏菌致病机制中的作用。该项目将建立在SM组的初步数据和现有工具(菌株和质粒)、SP组在沙门氏菌发病机制方面的专业知识以及MS在农场动物沙门氏菌感染模型中的体内技能的基础上。

In this project we will examine, for the first time, a role for mechanosensitive channels in microbial pathogenesis. Salmonella enterica (Salmonella) is a food-borne pathogen associated with around 27 million cases of typhoid fever and almost 100 million cases of gastroenteritis in humans each year. The majority of Salmonella enterica serotypes can infect a wide range of vertebrate hosts, including food-producing animals, which act as key reservoirs of infection. In farm animals some serotypes cause systemic infections similar to typhoid fever that impair welfare and productivity. However, the molecular mechanisms enabling these bacteria to colonise their hosts and produce disease require further study.Mechanosensitive (MSC) channels are ubiquitous throughout life kingdoms1. They are gated by changes in membrane tension and in higher organisms are involved in processes such as hearing, balance and pain perception. In bacterial cells they are required to survive hypoosmotic shocks, such as transfer from a high salt to a low salt environment. In this situation, unless the MS channels open to release cell solutes, bacterial cells lyse and die. The MscL and MscS channels are the principal channels involved in this response. They have been extensively studied at the biochemical, structural and genetic level in Escherichia coli. Bacterial strains have multiple MscS family members, for example E. coli and Salmonella species have 6 members, including YnaI (STM1663). The family are related by their common core domain structure but are distinguished by additional domains, often of unknown function. This diversity of structure and associated potential for variations in function is not well understood. Two recent studies have suggested that one of these channels, YnaI, is required for host tissue colonization and/or pathogenesis during bacterial infections of farmed animals. Chaudhuri et al used transposon-directed insertion-site sequencing (TraDIS) of S. Typhimurium and identified genes in which transposons caused attenuation2. Multiple independent insertions in ynaI impaired intestinal colonisation in pigs, cattle and chickens. Transcription levels of ynaI are slightly increased within macrophages (http://tinyurl.com/HintonLabSalCom). In a separate study, a YnaI homolog in Campylobacter jejuni was found to be required for colonisation of chicks3. YnaI is less understood than the MscS channel but exhibits unique electrical and physiological characteristics. The YnaI channel has a low conductance (~2pA, MscS = 25pA) and in excised patches requires a high pressure to gate, close to that which would otherwise lyse a cell. The protein comprises 5 transmembrane spans, with TM3-5 closely related to MscS. TM5 is the pore-lining sequence and links to the extensive C-terminal cytoplasmic domain. The channel, as typical for MscS-related proteins, is a homoheptamer. A cryo-electron microscopy study revealed that the cytosolic domain of YnaI is structurally similar to that of MscS and suggested that TM1-2 of each subunit is tilted away from the remaining 3 TMs. A similar gating mechanism has been proposed as for MscS. This project will aim at understanding the role of YnaI in Salmonella pathogenesis. The project will build on preliminary data and existing tools (strains and plasmids) from the SM group, the expertise of the SP group in Salmonella pathogenesis, and the in vivo skills of MS in Salmonella infection models in farm animals.