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

Lead Research Organisation: University of Aberdeen
Department Name: College of Life Sci and Med Graduate Sch

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M010996/1 01/10/2015 30/09/2023
1802201 Studentship BB/M010996/1 01/10/2016 30/09/2020 Mimi Nwakaego Asogwa
 
Description YnaI is one of the mechanosensitive channels found amongst many bacterial species including Salmonella Typhimurium. To investigate S. Typhimurium YnaI structure and function, the Salmonella Typhimurium ynaI was cloned into a plasmid, followed by physiological characterization of the Salmonella Typhimurium YnaI constructs expressed in an E. coli channel-less mutant strain. The Salmonella Typhimurium YnaI channel has unique characteristics when expressed in E. coli:
1. S. Typhimurium YnaI channel conferred protection against osmotic shock when overexpressed:
2. High level expression of S. Typhimurium YnaI inhibited growth in two different complex media and in minimal media.
3. When induced, Salmonella YnaI protein is abundantly expressed.

To understand the contribution of specific motifs or domains to YnaI channel opening, I am currently performing site-directed mutagenesis (to mutate specific residues) on the YnaI clone to create mutants. This will be followed by physiological characterization of the mutant.
To further understand other properties of the YnaI channel, I plan to get training on patch clamp techniques so as to assess the electrophysiological properties (pressure sensitivity, conductance and open dwell time) of YnaI.

To further understand the role of YnaI in S. Typhimurium pathogenesis, Salmonella Typhimurium ynaI gene was deleted and the ability of mutant to survive and replicate in host cells was investigated. Deletion of ynaI from Salmonella Typhimurium from led to increased internalization in macrophages and epithelial cells. More replicate of these experiments is currently ongoing.

We now better understand the Salmonella YnaI channel and more experiments are currently ongoing to further understand features of the channel that makes it unique from other mechanosensitive channels found in Salmonella. Data derived from this study reveals novel characteristics of Salmonella Typhimurium YnaI which may provide insights into other functions of the Salmonella Typhimurium YnaI channel.

I attended a Microbiology Society Conference in April, 2018 at Birmingham where I gave a poster presentation of my project and won the Journal of Medical Microbiology poster prize.
I have the opportunity to present my research project at various events, such as during our monthly joint lab meetings, at the University's annual symposium, the Eastbio annual symposium and the May festival in Aberdeen.

I have undergone a 3-month internship at NCIMB Ltd, Aberdeen and during that time I was involved in the development of metagenomic standard for Next generation sequencing. During that time:
Got a better understanding of the biodiversity of Marine bacteria.
Learnt to use instruments such as qPCR and 3 different sequencing platforms (Nanopore, Miseq and genetic analyser).
I got a better unsderstanding on customer expectation of microbiome standads and techniques as well as challenges faced by microbiome researches. The project I started has a prospect of developing into a future commercial product and I have expanded my network of contacts through interaction with a range of non-academic professionals.
Exploitation Route Understanding the role of YnaI in Salmonella infections will enhance understanding of mechanisms used by Salmonella in hosts colonization and disesase.
it will also provide opportunity to crete new stratagies to reduce food contamination and untimately the incidence of food-borne infections in humans.
Sectors Agriculture, Food and Drink