Development of probiotic-based and Clostridioides difficile-targeted therapeutics

Abstract Clostridioides difficile is a Gram-positive, spore-forming and toxin-producing anaerobic bacterium. It is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of life-threatening pseudomembranous colitis. Central to predisposition to C. difficile infection (CDI) is the disruption of the gut microbiota by broad-spectrum antibiotics. Currently, standard treatment of CDI is the administraion of vancomycin, fidaomicin or metronidazole, but none of these is fully effective as the antibiotic treatment results in an estimated 15-35% of recurrence. Treatment of recurrent CDI (rCDI) is one of the major challenges in the field. Alarmingly, significantly decreased susceptibility to metronidazole and vancomycin has been reported. As such, metronidazole is no longer recommeded as the first-line drug for CDI treatment. Novel non-antibiotic therapeutics that specifically target C. difficile are desperately needed to control CDI and recurrence. We have recently identified a C. difficile phage lysin lytic domain, designated as LCD, which potently and broadly lyse different ribotypes of C. difficile clinical isolates. We have also identified a panel of single-domain variable fragments of heavy-chain only antibodies (VHHs) against the unique and conserved C. difficile protein Cwp84. Cwp84 is a surface-associated cysteine protease and plays a critical role in the maturation of surface-layer proteins that are important for bacterial colonization, as antibodies against Cwp84 protected hamsters from lethal CDI by delaying C. difficile colonization. To generate a targeted therapy specifically against C. difficile, we have fused LCD with one anti-Cwp84 VHH, generating a novel fusion protein LCD-VHH27. LCD-VHH27 is significantly (p<0.0001) more potent than LCD in lysing different C. difficile strains including 2 hypervirulent epidemic RT027 strains. In this R21, we will generate more LCD-VHH fusions and use our novel antibiotic-free probiotic systems Saccharomyces boulardii and Lactococcus lactis to deliver the fusion proteins to the site of C. difficile colonization and infection in animal models of CDI. We hypothesize that engineered probiotics that secrete LCD-VHH fusions at the lower intestines where C. difficile colonizes will lead to a potent therapeutic efficacy against CDI.