Abstract
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.
Highlights
Clostridium difficile is a spore-forming, Gram-positive obligate anaerobe that is the most common cause of nosocomial infections in the United States [1]
Due to the highly reactive nature of heme, elevated concentrations are toxic to bacteria
These results present a mechanism employed by C. difficile to sense and reduce intracellular heme concentrations to relieve toxicity
Summary
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 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]. In order to defend against the stresses of heme-mediated damage, bacteria encode systems for heme sensing and detoxification [15,16,17,18,19,20,21]. In the Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis, the heme stress response is controlled by the heme sensing two-component system, HssRS, which regulates transcription of the ABC transporter HrtAB to reduce heme toxicity through efflux [15, 16, 22]. C. difficile does not contain orthologs of known heme detoxification systems, and it is unknown if this organism encounters heme during infection
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