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

在这个项目中，我们将首次研究机械敏感通道在微生物发病机制中的作用。肠道沙门氏菌（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，包括食品生产动物，它们是感染的主要宿主。在农场动物中，一些血清型引起类似于伤寒的全身感染，损害福利和生产力。然而，使这些细菌能够定植于宿主并产生疾病的分子机制需要进一步研究。它们通过膜张力的变化来门控，并且在高等生物中涉及听觉，平衡和疼痛感知等过程。在细菌细胞中，它们需要在低渗休克中存活，例如从高盐环境转移到低盐环境。在这种情况下，除非MS通道打开以释放细胞溶质，否则细菌细胞溶解并死亡。MscL和MscS通道是参与这种反应的主要通道。它们在大肠杆菌中的生化、结构和遗传水平上被广泛研究。细菌菌株具有多个MscS家族成员，例如E.大肠杆菌和沙门氏菌属有6个成员，包括Yna I（STM 1663）。该家族通过其共同的核心结构域结构而相关，但通过通常功能未知的额外结构域来区分。这种结构的多样性和相关的功能变化潜力还没有得到很好的理解。最近的两项研究表明，这些通道之一，YnaI，需要宿主组织定植和/或致病过程中的细菌感染的养殖动物。Chaudhuri等人使用转座子定向插入位点测序（TraDIS）的S。鼠伤寒沙门氏菌，并确定了转座子引起衰减的基因2。在猪、牛和鸡中，ynaI的多个独立插入损害了肠道定植。巨噬细胞内ynaI的转录水平略有增加（http：//tinyurl.com/HintonLabSalCom）。在另一项研究中，发现空肠弯曲菌中的YnaI同源物是鸡定殖所需的3。YnaI比MscS通道了解得少，但具有独特的电和生理特性。YnaI通道具有低电导率（~ 2 pA，MscS = 25 pA），并且在切除的斑块中需要高压来门控，接近于否则将裂解细胞的压力。该蛋白包含5个跨膜跨度，其中TM 3 -5与MscS密切相关。TM 5是孔衬里序列，并连接到广泛的C-末端胞质结构域。该通道，作为典型的MSC相关蛋白，是一个同源七聚体。冷冻电子显微镜研究表明，YnaI的胞质结构域的结构相似的MscS，并建议，TM 1 -2的每个亚基是倾斜远离其余3 TM。类似的门控机制已经被提出用于MscS。该项目旨在了解YnaI在沙门氏菌发病机制中的作用。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理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.