The role of central metabolism in the successful infection of macrophages and mice by Salmonella Typhimurium

The bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) is responsible for disease in animals and man. It causes gastroenteritis in humans characterised by stomach ache and bloody diarrhoea. S. Typhimurium is responsible for up to 20 million cases of gastroenteritis each year worldwide. In the USA and UK, Salmonella causes more human deaths than any other food-borne pathogen. The process of infection starts when contaminated food or water is ingested. S. Typhimurium bacteria travel to the intestine where they invade the cells which line the gut wall (epithelial cells). The bacteria then break out of the epithelial cells, causing bloody diarrhoea, and invade immune cells which are responsible for fighting infection (macrophages). Although the macrophages are designed to kill bacteria, S. Typhimurium has developed the ability to evade the chemical weapons deployed by the macrophage to kill it. S. Typhimurium does this by constructing a protective region within the macrophage, the Salmonella Containing Vacuole or SCV. The Salmonella survive and grow inside the macrophages which carried around the body, giving the bacteria the opportunity to infect other organs including the lymph nodes, spleen and liver. We don't know very much about the environment inside the SCV and are particularly interested in which chemical compounds are available for Salmonella to use as fuel, providing the bacteria with the energy it needs to survive and grow. Salmonella can use a variety of different chemicals as fuel, but in the same way we need different car engines to use petrol or diesel, Salmonella needs to make different cellular machines (enzymes and transport proteins) to use the different chemicals efficiently. The transport proteins bring fuel chemicals into the Salmonella cells and different sets of enzymes act together to form pathways which speed up breakdown of the fuel chemicals. This means if we know which transport proteins and enzymes the bacteria are making we can get clues about which fuel chemicals the bacteria are using to grow. The transport proteins and enzymes made in the cell are determined by the presence of RNA molecules which act as messenger signals and tell the cell which particular transport proteins and enzymes to make. By using a special technique called transcriptomics to look at which RNA molecules are present in the cell, we can tell which transport proteins and enzymes are likely to be produced. We already have transcriptomics data from Salmonella during infection which suggests that the potential fuel sources for Salmonella inside macrophages include sugars and fats. This proposal aims to determine whether sugars and fats are actually used as fuel by the Salmonella during infection. We will do this by blocking the manufacture of specific enzymes and transport proteins involved in the fuel breakdown pathways and seeing whether this reduces the ability of Salmonella to survive inside the SCV. We will find out whether the same fuel or different types of fuel are used during infection. Macrophages also make a chemical called interferon that stimulates the breakdown pathways of fat in other species of bacteria that live inside macrophages. When these pathways are blocked by stopping specific enzymes involved in the pathway from being produced, the survival of bacteria reduces. We aim to find out whether interferon stimulates similar breakdown pathways in Salmonella, and whether blocking these pathways also reduces infection. Identification of the breakdown pathways and the chemicals used by Salmonella to survive inside the macrophage is likely to suggest ways of preventing Salmonella infections.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major pathogen of animals and man, causing at least 20 million cases of gastroenteritis each year in both industrial and developing nations. In the US and UK, Salmonella causes the majority of foodborne illness-related human deaths. Although S. Typhimurium infections cause gastroenteritis in humans, in mice they result in systemic disease which has been used as a model for typhoid fever. S. Typhimurium infections are usually acquired by ingestion of contaminated food or water. In systemic (typhoid-like) disease, the bacteria pass through the stomach and colonise the Peyer's patches of the small intestine. S. Typhimurium penetrates the small intestinal barrier by selectively invading M cells, specialised antigen-sampling epithelial cells. From there the Salmonella disseminate to the local mesenteric lymph nodes and then to the spleen and liver via phagocytic cells such as macrophages, dendritic cells and neutrophils. Once inside macrophages the Salmonella reside in a specialised acidic compartment, known as the Salmonella Containing Vacuole (SCV). The SCV acts as a shield, preventing lysosomal fusion and protects bacteria from attack by innate host cell defence mechanisms. In order to survive and replicate within macrophages, the Salmonella must adapt to utilise the limited carbon sources and other nutrients available within the SCV. For other pathogenic bacteria studied during intracellular growth these metabolic adaptations seem to be intimately linked with not only virulence but also with the immune status of the macrophage. Using a microarray-based approach, the Molecular Microbiology group at the IFR have identified the expression levels of all the S. Typhimurium genes transcribed during infection of macrophages. These data have permitted the identification of genes encoding enzymes involved in central metabolism that show differential expression during infection. Our findings have led us to hypothesise that specific nutrients sustain the growth of intracellular S. Typhimurium within the SCV. We will test our hypothesis by deleting key genes involved in the transport and metabolism of potential nutrients utilised by intracellular S. Typhimurium. The mutant strains will be used in a series of infection experiments to determine their ability to invade and persist within macrophages grown in vitro and during systemic infection of mice. We will also perform enzyme assays on extracts of S. Typhimurium that have been isolated from infected non-activated and activated macrophages, and SCV's. These experiments will define which nutrients and metabolic pathways are utilised by intracellular S. Typhimurium and whether these play a role in virulence. Such information will be completely novel for S. Typhimurium. We will construct fluorescent reporter gene fusions to metabolic genes from catabolic pathways used by intracellular S. Typhimurium to determine their relative importance and time of induction during infection of macrophages. The fusions will also be used to determine the effect of macrophage activation on expression of key catabolic enzymes used by intracellular S. Typhimurium. Our novel approach promises to improve our understanding of the infection biology of S. Typhimurium.