The Role of Central Metabolism in the Successful Infection of Macrophages and Mice by Salmonella Typhimurium

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. <P>
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. <P>
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. <P>Our novel approach promises to improve our understanding of the infection biology of S. Typhimurium.