The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice

Ferrous iron is the dominant form of iron in the oxygen-limited large intestine, the site for Clostridioides difficile infection (CDI). We investigated the extent to which C. difficile requires the ferrous iron transporter 1 (FeoB1), a main permease for iron acquisition, using in vitro and in vivo approaches. Construction of feoB1 deletion mutant in C. difficile R20291 (i.e., R20291{Delta}feoB1) decreased intracellular iron content by [~]25%. This was accompanied by downregulation of tcdA and tcdB genes and reduced synthesis of TcdA and TcdB toxins, which was reflected by a [~]1000-fold reduction in cytopathy against Vero cells. Complementation with WT feoB1 restored toxin production. Transcriptional and biochemical analyses revealed {Delta}feoB1 altered metabolic pathways that negatively impact toxin production, including downregulation of the oxidative branch of the Kreb cycle and an associated cellular accumulation of pyruvate. Hence FeoB1 influences multiple bioenergetic and redox pathways, which in turn inhibits toxin biosynthesis. R20291{Delta}feoB1 was avirulent in mice with underlying colitis. In this model, mice are prone to develop severe CDI, but mice infected with the mutant lacked diarrheal symptoms, had lesser inflammatory responses, bacterial bioburdens and cecal toxin titers. These results strongly imply that ferrous iron acquisition and homeostasis is central to toxin metabolism and virulence. Hence targeting FeoB1 could be a potential therapeutic strategy to impede the colonization and pathogenesis of C. difficile, even in the setting of colitis where intestinal bleeding may occur and CDI is more severe.