Impact of Magnesium on Salmonella Infection and Therapy

The focus of this project is the role of magnesium in Salmonella infections at the molecular, cellular, tissue, and organismal levels. The results will reveal mechanisms underlying a novel paradigm for nutritional immunity, and might open avenues for strengthening and synergizing with host immunity to control infections.Bacterial infections are a major threat for human health. Development of urgently needed novel antimicrobials is slow. Strengthening host immunity is an attractive alternative approach. One critical component of immunity is to deprive pathogens of essential nutrients, thereby blocking their growth and virulence ("nutritional immunity"). Nutritional immunity is best characterized for transition metals including iron, manganese, and zinc.This project focuses on Salmonella enterica serovar Typhimurium infections in its natural host, the mouse. This infection model is extensively characterized because of facile pathogen and host genetics, reproducible infections with comparatively small inocula, and the availability of diverse tools and techniques that were developed by numerous research groups for this model. Invasive salmonellosis in mice reproduces some aspects of human salmonellosis which causes major morbidity and mortality. The increasing drug resistance of Salmonella has led to its recognition as a WHO priority pathogen.We have shown that Salmonella struggle to obtain sufficient Mg2+ for replication in mice because of the major host resistance factor SLC11A1 (NRAMP1). In particular, inactivating point mutations in Salmonella high-affinity Mg2+ slow replication in mice with, but not without, functional SLC11A1. Magnesium is essential for key aspects of Salmonella physiology including envelope integrity, ribosomes, DNA, RNA many enzymes, and metabolites such as adenosine triphosphate. However, key questions about the role of magnesium during infection remain open: How does SLC11A1 sequester Mg2+? Does Salmonella's own growth diminish Mg2+ levels in its microenvironment in host vacuoles? Why is Mg2+ starvation heterogeneous across the Salmonella population? How can dietary Mg2+ influence Salmonella replication, inflammation, and the overall course of disease? How does SLC11A1 impact on the course and outcome of antimicrobial treatment? We address these questions by testing four hypotheses:(1) SLC11A1 variants with abolished Mg2+ transport fail to confer resistance.(2) Multiple Salmonella residing together in the same host cell vacuole compete for Mg2+ resulting in exacerbated Mg2+-starvation and diminished replication.(3) Salmonella access to magnesium is particularly poor at advanced stages of infection, close to inflammation foci, and during hypomagnesemia.(4) SLC11A1 delays antibiotic clearance of Salmonella but enables sustained control of surviving Salmonella after treatment termination.To test these hypotheses, we employ population and single-cell approaches including Salmonella mutants with specific metal-uptake defects, metal-responsive reporter strains, ex-vivo proteomics, high-throughput feedback microscopy, immunohistochemistry, and detection of individual Salmonella cells in whole organs using serial two-photon tomography. These methods are well established in our laboratory and have sufficient statistical power to detect the expected effects.The results will reveal mechanisms underlying magnesium-based nutritional immunity against Salmonella. This will provide exciting opportunities to compare the novel paradigm of the main group metal magnesium to extensively characterized transition metals as substrates for nutritional immunity. The insights into the link between inflammation and nutritional immunity as well as responses to antimicrobial chemotherapy might open novel avenues for strengthening and synergizing with host immunity for improved infection control.