CHAP^SAP26-161, A Truncated Protein and Enzymatic Active Domain of Endolysin LysSAP26, as a Potential Therapeutic Agent to Combat Clostridioides difficile Infection

Clostridioides difficile is a spore-forming enteric pathogen that causes life-threatening diarrhea and colitis. Notably, C. difficile infection (CDI) is a major healthcare-associated infection with increasing incidence and morbidity rates. Antibiotic-induced microbial disruption has been linked to susceptibility to CDI transmission and relapse. Therefore, alternative therapeutic approaches that effectively prevent C. difficile growth and spore germination are urgently needed. Bacteriophage-derived endolysins and their derivatives have recently shown potential as novel antibacterial agents. Hence, this study aimed to investigate the efficacy of a novel truncated cysteine-histidine-dependent amidohydrolase/peptidase (CHAP) modular endolysin, CHAP^SAP26-161, in combating CDI. In vitro studies demonstrated its potent bactericidal activity against several clinically relevant C. difficile strains, including toxin A-and toxin B-producing and nontoxigenic strains. CHAP^SAP26-161 exhibited rapid and specific killing activity, thereby significantly reducing C. difficile colony-forming units. Furthermore, in a murine CDI model, CHAP^SAP26-161 treatment remarkably reduced C. difficile burden and clinical symptoms, such as diarrhea and weight loss. In histopathological analysis, colonic inflammation and tissue damage decreased in CHAP^SAP26-161-treated mice compared with that in the control group. More-over, no cytotoxic effects were observed on the A549 cell line, indicating that CHAP^SAP26-161 is safe as a therapeutic agent. These findings highlight that CHAP^SAP26-161 is a promising treatment option for CDI. Importantly, preclinical and clinical studies are warranted to fully evaluate the therapeutic potential of CHAP^SAP26-161 and its possible implementation in clinical practice.