The SitABCD, MntH and ZupT Transporters of S. Typhimurium Promote Evasion of Calprotectin-Mediated Manganese Sequestration and Colonization of the Inflamed Gut

The pathogen Salmonella Typhimurium causes gastroenteritis, characterized by intestinal inflammation and diarrhea. S. Typhimurium thrives in the inflamed gut because it acquires nutrients, including essential metal ions like iron and zinc. Manganese (Mn²⁺) is another metal ion that contributes to S. Typhimurium growth in systemic infections, however, it is not known whether Mn²⁺ acquisition also promotes Salmonella colonization of the inflamed gut. To test this, we constructed S. Typhimurium mutants in the two major Mn²⁺ transporters SitABCD and MntH and a broad divalent cation transporter ZupT. We then infected mice with a 1:1 mixture of wild-type S. Typhimurium to mutants in these transporters and we measured colony forming units (CFU) from fecal pellets collected 48, 72 and 96 hours post-infection (p.i.) as a proxy for colonization of the large intestine. We found that wild-type S. Typhimurium significantly outgrew both the sitA mutant and the mntH mutant up to 50-60 fold. Furthermore, wild-type S. Typhimurium also significantly outgrew a mutant lacking sitA and mntH as well as a mutant in sitA, mntH, and zupT, up to 400 fold. Next, we set out to determine the mechanism by which SitABCD, MntH, and ZupT promote S. Typhimurium growth in the inflamed gut. We hypothesized that these transporters facilitate evasion of manganese starvation caused by the host antimicrobial protein calprotectin, which is highly induced in the inflamed gut during S. Typhimurium infection. We thus tested whether SitABCD, MntH and ZupT promoted S. Typhimurium growth in vitro in the presence of physiologically relevant concentrations of calprotectin (250 μg/ml). While wild-type S. Typhimurium grew well in media supplemented with calprotectin, the sitA mntH zupT mutant was strikingly inhibited, and no colonies were recovered at 16 hours post-inoculation. Notably, growth of the sitA mntH zupT mutant was comparable to wild-type growth in the presence of calprotectin mutants in the manganese-binding site. Because calprotectin has a marked antimicrobial activity in vitro, we next tested whether the growth defect of the sitA and sitA mntH mutants could be rescued in calprotectin-mutant mice. However, we only observed partial rescue of the sitA mutant, while the sitA mntH mutant was not rescued, indicating that other proteins may synergize with calprotectin in vivo and sequester manganese in the gut. Overall, our data demonstrate that SitABCD, MntH, and ZupT promote S. Typhimurium colonization of the inflamed cecum and evade calprotectin-mediated manganese sequestration. Furthermore, we found that manganese binding is essential for the antimicrobial activity of calprotectin against S. Typhimurium. However, other host factors in addition to calprotectin may sequester manganese and further indicate that this metal is important for Salmonella colonization of the inflamed gut and competition for a niche.