Project Summary Chemical Biological Discovery of Lipid Virulence Factors in the Major Bacterial Pathogens For decades, the search for the causes of bacterial virulence has focused on genes rather than metabolites. Genetic approaches have been broadly successful, and modern infectious disease research relies fundamentally on genomic maps of the major pathogens. Owing to the lack of whole-organism chemical biology tools, bacterial lipids have not been systematically tested for their roles in virulence, even though lipids are the primary interface with the human host, where they control nutrient flow and trigger host immune response. We invented a mass spectrometry platform for lipid profiling to detect nearly all ionizable lipids in a bacterial cell within 2 hours. Experiments on Mycobacterium tuberculosis and Salmonella enterica serovar Typhi now provide clear evidence for the general insight that many, perhaps the majority, of lipids in the world's bacterial pathogens, are currently unknown as named compounds. Based on successes in identifying virulence factors in two major pathogens of worldwide significance, we will carry out a chemical biology approach known as forward lipidomics. Specifically, we will use whole organism mass spectrometry profiling to discover the lipids that are selectively expressed in virulent bacteria and are unknown in existing lipid catalogs. Then, we will chemically synthesize the virulence associated lipids and link them to their biosynthetic genes for deletion in bacteria using reverse genetic approaches. Using genetically modified bacteria that are deficient in defined lipids, we will determine the roles of virulence lipids during infection. Using nature identical synthetic lipids, we will determine the cellular mechanisms of generation of foamy macrophages and identify immune receptors that mediate host response. We will create lipid maps of the major Gram negative pathogen groups based on patient strains to build the overlooked field of chemical biology of bacterial virulence. These basic and translational studies will support the development new forward lipidomics approaches to the diagnosis and treatment of major infectious diseases.