Research Article: Heme sensing and detoxification by HatRT contributes to pathogenesis during Clostridium difficile infection

Date Published: December 21, 2018

Publisher: Public Library of Science

Author(s): Reece J. Knippel, Joseph P. Zackular, Jessica L. Moore, Arianna I. Celis, Andy Weiss, M. Kay Washington, Jennifer L. DuBois, Richard M. Caprioli, Eric P. Skaar, Theresa M. Koehler.


Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that infects the colon, causing symptoms ranging from infectious diarrhea to fulminant colitis. In the last decade, the number of C. difficile infections has dramatically risen, making it the leading cause of reported hospital acquired infection in the United States. Bacterial toxins produced during C. difficile infection (CDI) damage host epithelial cells, releasing erythrocytes and heme into the gastrointestinal lumen. The reactive nature of heme can lead to toxicity through membrane disruption, membrane protein and lipid oxidation, and DNA damage. Here we demonstrate that C. difficile detoxifies excess heme to achieve full virulence within the gastrointestinal lumen during infection, and that this detoxification occurs through the heme-responsive expression of the heme activated transporter system (HatRT). Heme-dependent transcriptional activation of hatRT was discovered through an RNA-sequencing analysis of C. difficile grown in the presence of a sub-toxic concentration of heme. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). Strains inactivated for hatR or hatT are more sensitive to heme toxicity than wild-type. HatR binds heme, which relieves the repression of the hatRT operon, whereas HatT functions as a heme efflux pump. In a murine model of CDI, a strain inactivated for hatT displayed lower pathogenicity in a toxin-independent manner. Taken together, these data suggest that HatR senses intracellular heme concentrations leading to increased expression of the hatRT operon and subsequent heme efflux by HatT during infection. These results describe a mechanism employed by C. difficile to relieve heme toxicity within the host, and set the stage for the development of therapeutic interventions to target this bacterial-specific system.

Partial Text

Clostridium difficile is a spore-forming, Gram-positive obligate anaerobe that is the most common cause of nosocomial infections in the United States [1]. C. difficile infects the colon, causing a wide range of diseases that vary from infectious diarrhea to pseudomembranous colitis. During infection, C. difficile produces two potent toxins, TcdA and TcdB, which cause severe damage to intestinal epithelial cells resulting in inflammation, fluid secretion, and necrotic cell death [2, 3].

C. difficile infection of the colon causes severe epithelial cell damage, inflammation, and edema, which leads to the hallmarks of C. difficile-colitis. Importantly, this damage and subsequent inflammatory response also creates a hostile environment for bacteria within the gut [2, 31–33]. Highly reactive heme molecules that can be toxic to bacteria are released into the lumen through erythrocyte lysis and necrotic epithelial cell death [3, 5]. Despite the hazard of heme toxicity, C. difficile thrives in the colon and survives in the presence of high heme levels. Prior to this work, the mechanism by which C. difficile resists heme toxicity were unknown. Herein, we visualized the high abundance of hemoglobin during infection, serving as a proxy for heme, in the murine ceca during CDI. We identified a molecular mechanism encoded by the hatRT operon to sense and detoxify heme in C. difficile. HatR functions as a transcriptional repressor of the hatRT operon and responds to heme concentrations through direct binding of heme. HatR-heme complexes de-repress the hatRT operon, leading to the HatT-mediated reduction in intracellular heme concentrations, presumably through efflux. In support of these data, strains with inactivated hatR or hatT exhibited delayed growth in the presence of heme and the hatT::CT strain conferred reduced pathology in a toxin-independent manner in a mouse model of CDI.




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