The importance of the intracellular energy metabolism of Salmonella Typhimurium within epithelial cells and macrophages

According to the latest European Food Standard Agency (EFSA) statistics (2008), Salmonella enterica serovar Typhimurium (S. Typhimurium) is the second most reported zoonotic infection in humans and the most frequent cause of food borne outbreaks in the EU. Worldwide Salmonella is responsible for up to 800,000 deaths from contaminated food and water. Following ingestion Salmonella bacteria travel to the intestine where they invade the cells lining the gut wall (epithelial cells), causing bloody diarrhoea. In the case of systemic infections (caused by S. Typhi and S. Paratyphi in humans), Salmonella invade the immune cells which are responsible for fighting infection (macrophages). Although the role of macrophages is to kill bacteria, Salmonella has adapted to evade the lethal chemical weapons deployed by macrophages. The Salmonella are able to survive and grow within the macrophages in a specialised compartment known as the 'Salmonella containing vacuole' (SCV), and thereby become systemically disseminated to other organs including the mesenteric lymph nodes, liver and spleen. We recently made the discovery that the major metabolic pathway required to catabolise sugars (glycolysis) is essential for the ability of Salmonella to grow and survive within macrophages, but not within epithelial cells. In order to grow and survive within host cells, Salmonella must also have a route for generating the energy required for these processes. Together with other evidence from our research, and published data, it seems likely that S. Typhimurium generates energy via different mechanisms in macrophages compared to epithelial cells. One of the aims of this proposal is to differentiate between these alternative energy generating pathways in macrophages and epithelial cells. Such information may facilitate therapeutic interventions. One of the major questions in infection biology is the extent to which the host cell contributes to the intracellular growth of Salmonella. We will use cutting edge techniques to determine the contribution of amino acids derived from host proteins and peptides to the intracellular growth of Salmonella in infected epithelial cells and macrophages. If Salmonella is dependent on the host for some of its requirements to enable intracellular growth, then this may also represent a way to facilitate therapeutic intervention.

In humans S. Typhimurium causes a severe self-limited gastroenteritis, however in mice it becomes systemic resulting in typhoid like fever. In order to become systemic, S. Typhimurium first colonises the specialised antigen sampling epithelial cells lining the Peter's patches of the small intestine, then invades phagocytic cells such as macrophages in the mesenteric lymph nodes. Within macrophages, Salmonella resides within a specialised compartment (the Salmonella containing vacuole (SCV). We recently showed that glycolysis and glucose are required for Salmonella to replicate and survive in macrophages. However, this and other data from our group suggests Salmonella generates ATP for replication via different mechanisms in macrophages and epithelial cells. We hypothesise that S. Typhimurium generates ATP via oxidative phosphorylation in infected epithelial cells, but has to resort to substrate level phosphorylation for ATP generation in macrophages. We also hypothesise that the necessity to use substrate level phosphorylation for ATP generation in infected macrophages is a consequence of the inhibition of the electron transport chain due to defensive reactive oxygen species produced by the host. The mode of energy generation for intracellular growth of Salmonella may be important since the syntheses of virulence determinants, such as flagella and secretion apparatus in intracellular Salmonella are ATP-dependent. In this proposal we will also address a major question that has proved difficult to answer in the past: to what extent does the host contribute to replication and survival of Salmonella within the SCV? We will use a novel application of a proteomic approach (stable-isotope labelling by amino acids in cell culture - SILAC) to determine the contribution of amino acids from host proteins and peptides to the replication of S. Typhimurium within macrophage and epithelial cells

Salmonella is a major cause of food-borne bacterial deaths in the UK, and outcomes from this project may contribute to underpinning strategically-driven research to reduce the burden of food borne disease in the UK, and to enhance the economic viability of the UK farming industry. Particularly worrying is the emergence of antibiotic resistant strains of Salmonella over the preceding decade. Defining the energy generating mechanisms and the contribution of the host to the intracellular replication of Salmonella may facilitate the development of vaccine strains or therapeutic intervention strategies that will address the emerging future problem of multi-antibiotic resistant strains. The novel application of the SILAC technique described in this proposal could also be extended to identify differences in metabolism-related proteins between ubiquitous and host restricted and adapted serovars during intracellular metabolism in animal host cell lines which would help to address the long standing question of which factors contribute to host specificity. Further funding would be sought for this work. On a more general note, the results from the proposed research project may contribute to knowledge-led approaches for improved food safety in the UK, thus addressing Policy priorities of the BBSRC, and DEFRA, FSA and EU-driven aims of reducing infection of food animals with food borne pathogens. The outcomes of the research will have the long-term potential to shape public policy and create future economic and social impact by reducing Salmonella contamination of the food chain and environment. The impact of the proposed research will be conveyed to interested parties with the support of the Communications team at IFR and Media Department of BBSRC. The IFR maintains a website on research activities at IFR and information on IFR science is disseminated through the IFR Science & Innovation newsletter. We provide independent advice to the general public and government, which inform and affect government policy. IFR hosts informative websites such as Food Databanks, ComBase and Food Allergy Information. The IFR maintains well established strategies for communicating its research via several routes. These include (1) peer-reviewed scientific publications (2) presentations at scientific conferences (3) general presentations to the wider scientific community and the public via newsletters, press releases, and websites. We will also work in collaboration with the Communications teams and others to maximize utilization of outreach opportunities, via public showcases and participation in public events.