Salmonella enterica is the leading cause of bacterial food borne enteritis and the leading cause of death from food borne disease in the United States. Salmonella Typhimurium (STm) encodes at least 11 multidrug efflux systems (MES), known to export antibiotics and a few other substrates from the cell, with high redundancy in substrate specificity for antibiotics. Mutants lacking MacAB, an ABC-type MES known to weakly efflux macrolide antibiotics, are avirulent in animal models suggesting an important role in infection. In published work we have shown that MacAB protects STm from peroxide mediated killing by secreting a heat stable compound that detoxifies extracellular reactive oxygen species (ROS). ROS are antimicrobial compounds produced in abundance by host neutrophils and macrophages in response to STm and other bacterial infections. Our unpublished genetic evidence strongly suggest that one or more linear metabolite(s) of enterobactin (DHBS3, DHBS2, DHBS) are likely the natural antioxidant substrates secreted by MacAB that protect Salmonella against ROS. Mutants that cannot produce DHBS trimers, dimers or monomers share the phenotypes of a macAB mutant during ROS exposure, and cannot rescue the macAB mutant from peroxide mediated killing when co-cultured. Although the outer membrane partner for MacAB function during macrolide efflux is TolC, this protein does not appear to function with MacAB for secretion of antioxidant compounds during oxidative stress. Two other putative outer membrane proteins, STM2172 and STM2690, share peroxide sensitivity of macAB mutants during oxidative stress when these outer membrane proteins are deleted together, and the double mutant fails to rescue a macAB mutant from ROS mediated killing. In order to develop a molecular and mechanistic understanding of the function and components of the MacAB system in H2O2 resistance, we have developed two hypotheses addressed in two specific aims. First, (Aim I) we hypothesize that linear products of enterobactin (DHBS3, DHBS2, DHBS) are the substrates of the MacAB system that detoxify peroxide. Second, (Aim II) we hypothesize that STM2172 and STM2690 are the outer membrane components that complete the MacAB apparatus allowing export of the substrate out of the bacterium. We are uniquely positioned to address these hypotheses because we possess critical preliminary data, expertise and the tools necessary to complete the work we propose. Our studies described here on MacAB, and our long-term goal to understand the natural functions of multi-drug efflux systems, address a key gap in our understanding bacterial efflux pumps: their natural role in the biology of these organisms. Finally, a detailed understanding of MacAB function, and MES function more broadly, will provide new targets for the development of novel antimicrobial therapy.
Salmonella is a leading cause of food borne illness, causing ~1.4 million cases of diarrheal disease per year and is the single most common cause of death from food-borne illnesses associated with viruses, parasites or bacteria in the US primarily in immunocompromised persons. The genes and mechanisms used by non-typhoidal Salmonellae to survive in the intestine during gastroenteritis are not well understood. Development of a better understanding of the genes and mechanisms involved in this important stage of infection is critical to breaking the cycle of transmission of this organism. The work in this proposal will hav a direct impact on public health by providing novel targets for the development of new therapies and vaccines to reduce the burden of Salmonella infection in humans and in livestock.
