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

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.