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

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology


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.

Technical Summary

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

Planned Impact

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.
Description Salmonella is an enteric pathogen responsible for a variety of disease outcomes in humans and animals ranging from self-limited gastroenteritis to lethal typhoid fever. It is estimated that worldwide, typhoidal and non-typhoidal Salmonella infections result in an estimated 20 and 98.3 million human cases each year, of which 200,000 and 155,00 result in death respectively. One of the most intriguing and highly relevant questions regarding Salmonella infection is the metabolic adaptations required to enable intracellular replication of Salmonella bacteria within host cells, and how these contribute to the overall pathogenicity of the organism. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted a key glycolytic gene, pfkA to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is the major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells. In a second area of the project, we used murine epithelial and murine and human macrophage cell lines. The epithelial cell lines were mICc12, a transimmortalised murine colon enterocyte cell line that shows many of the characteristics of a primary epithelial cell line. The macrophage cell lines were Thp-1 human macrophages and RAW 264.7 murine macrophages. We used a mutational approach combined with an exometabolomic analysis to show that neither fermentative metabolism nor anaerobic respiration play major roles in energy generation in any of the cell lines studied. Instead, we identified overflow metabolism to acetate and lactate as the foremost route by which S. Typhimurium fulfils its energy requirements.
Exploitation Route Knowing the pathways of glucose metabolism in vivo will help to identify possible sites of enzyme inhibition that would prevent replication of the bacteria
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology