Blocking Toxin function and Modulating Gut Microbiota: Caffeic Acid and its Derivatives as Potential Treatments for Clostridioides difficile Infections

Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea that seriously threaten public health. Disruption of normal gut microbiota by the use of broad-spectrum antimicrobial agents enables C. difficile to proliferate in the colon. The emergence and prevalence of hypervirulent C. difficile strains result in increased morbidity, mortality and recurrence rates of CDI, thus creating a pressing need for novel therapeutics. The multi-domain toxins TcdA and TcdB are the primary determinant of CDI pathogenesis, renders them ideal drug targets in the anti-virulence paradigm. In this study, we identified caffeic acid and its derivatives as active inhibitors of TcdB via a cell-based high-throughput phenotypic screening. Further mechanistic investigations revealed that caffeic acid phenethyl ester (CAPE) could directly bind to TcdB, thus suppressing InsP6-induced autoproteolysis and inhibiting the glucosyltransferase activity. CAPE-treatment remarkably reduces the pathology of CDI in a murine infection model in terms of alleviated diarrhea symptom, decreased bacterial colonization and relieved histopathological lesions. Moreover, CAPE-treatment of C. difficile-challenged mice induces remarkable increase in the diversity and composition of the gut microbiota (e.g. Bacteroides) and alterations of gut metabolites (e.g. adenosine, D-proline and melatonin), which might partially contribute to the therapeutic outcomes of CAPE against CDI. Our results reveal the potential of CAPE as a therapeutic for the management of CDI, or CAPE might be served as a lead compound for the development of anti-virulence drug targeting TcdB.