Salmonella enterica serovar Typhimurium (S. Typhimurium) induces a host inflammatory response that suppresses the growth of commensal bacteria. A contributing factor is the expression of proteins that starve microbes of metals. One such protein, calprotectin (CP), sequesters zinc (Zn2+) and manganese (Mn2+). While growth of commensal bacteria is suppressed, intestinal inflammation enhances S. Typhimurium growth. We tested the hypothesis that Salmonella is resistant to CP-mediated Mn2+ starvation. Using wild-type CP or CP mutants deficient in either Zn2+ or Mn2+ binding, we determined that S. Typhimurium resisted CP-mediated Mn2+ starvation and growth inhibition by expressing three Mn2+ transporters (SitABCD, MntH, and ZupT) . We also show that Salmonella mutants lacking Mn2+ transporters had a growth defect in the inflamed gut that was not rescued in CP deficient (S100a9-/-) mice. Remarkably, growth inhibition of the sitA mntH zupT mutant was reduced in Il22-/- mice, which lack a key cytokine involved in the induction of metal-binding antimicrobials, a finding that suggests the host might also deploy CP-independent mechanisms of Mn2+ sequestration.

Under anaerobic conditions, CP did not inhibit sitA mntH zupT growth, suggesting that Mn2+ sequestration may enhance Salmonella susceptibility to oxidative stress. Consistent with this hypothesis, Mn2+ is a co-factor for SodA, a superoxide dismutase in Salmonella that reduces oxidative stress. We found that SodA helped S. Typhimurium overcome CP-mediated growth inhibition and contributed to Salmonella growth in the inflamed gut. Because neutrophils are the major source of oxidative stress during Salmonella induced gastroenteritis, we partially rescued the growth defect of the sitA mntH zupT and sodA mutants when we depleted neutrophils in C57BL/6 mice. We also rescued the growth defect of the sitA mntH zupT and sodA mutants in Cybb-/- mice, which have a defect in producing ROS. Moreover, the growth defect of the sitA mntH zupT mutant was less prominent in Cybb-/- S100a9-/- mice, most likely because these mice lack some ability to enhance oxidative stress through CP-mediated Mn2+ sequestration. Altogether, we demonstrate that Mn2+ transporters contribute to S. Typhimurium growth in the inflamed gut by helping Salmonella overcome Mn2+ starvation to effectively mitigate the host oxidative burst.