Heme Homeostasis and Utilization in Clostridioides difficle Pathogenesis and Persistence

Abstract

Invading microbial pathogens must sense and respond to the hostile environment of the vertebrate host to survive and cause infection. Clostridioides difficile, formerly Clostridium difficile, is the leading cause of hospital-acquired antibiotic-mediated diarrhea worldwide. Infection of the colon leads to severe inflammation and damage to the gastrointestinal epithelium due to the production of two potent toxins. Bacterial toxins produced during C. difficile infection 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, I 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) and the heme sensing membrane protein system (HsmRA) that senses and sequesters host heme to reduce heme-mediated toxicity. HatRT is comprised of a TetR family transcriptional regulator (hatR) and a major facilitator superfamily transporter (hatT). 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. HsmRA is comprised of a MarR-family transcriptional regulator (HsmR) and a membrane protein (HsmA). HsmR senses vertebrate heme, leading to increased expression of the hsmRA operon and subsequent deployment of HsmA to capture heme and reduce redox damage caused by inflammatory mediators of protection and antibiotic therapy. Strains with hsmR or hsmA inactivated have increased sensitivity to vancomycin and reduced colonization persistence in a murine model of relapse C. difficile infection. Taken together, these results define a mechanism exploited by C. difficile to repurpose and detoxify heme liberated within the inflamed gastrointestinal tract as a shield against antimicrobial compounds encountered at the host-pathogen interface, establishing HatT and HsmA as therapeutic targets for the development of treatments against C. difficile infection.