The Ferrous Iron Transporter FeoB1 is Essential for Clostridioides difficile Toxin Production and Pathogenesis in Mice
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ABSTRACTFerrous 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., R20291feoB1) decreased intracellular iron content by ~25%. This was accompanied by reduced synthesis of TcdA and TcdB toxins and downregulation of tcdA and tcdB genes, which was reflected by a ~1000-fold reduction in cytopathy against Vero cells. Complementation with WT feoB1 restored toxin production. R20291feoB1 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. Transcriptional and biochemical analyses revealed 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. Targeting FeoB1 could be a potential therapeutic strategy to impede the colonization and pathogenesis of C. difficile, even in the setting of colitis where CDI is more severe and intestinal bleeding may occur.AUTHOR SUMMARYClostridioides difficile is a major cause of antibiotic associated diarrhea that result in significant morbidity and mortality in hospitals. The ability of C. difficile to cause disease and damage the gut is directly related to the production of toxin A and toxin B. Without these toxins, C. difficile is not virulent. There is need to identify drug targets and therapeutic strategies that prevent C. difficile from producing toxins A and B. For C. difficile to colonize the large bowel, it must acquire ferrous iron, which is thought to be the main form of iron within the oxygen-limited large bowel environment. We show that genetic deletion of the ferrous iron transporter FeoB1 stops C. difficile from producing significant amounts of toxins under in vitro conditions. Importantly, the mutant R20291feoB1 was avirulent when tested in a mouse model that favors the development of severe infections. Avirulence of R20291feoB1 was related to the strain having significant metabolic changes that negatively impact toxin production. For example, cells could not properly metabolize pyruvate, which became accumulated in cells; pyruvate is known to reduce the production of toxins A and B. Our study provides new insights into how FeoB1 affects C. difficile colonization and disease development, indicating FeoB1 could be a drug target to stop toxin production in this bacterium.
