Bioengineering of bacteriophage-derived endolysin against Clostridioides difficile

Abstract

Clostridioides difficile is a significant cause of antibiotic-associateddiarrhea and pseudomembranous colitis. The extensive use of antibiotics drives the emergence of new antibiotic-resistant C. difficile strains with high virulence and low susceptibility to current antibiotic treatment. Therefore, the development of alternativetherapy is required. Phage endolysin is a peptidoglycan hydrolase enzyme potentially an antibacterial agent for bacterial infection treatment. To apply endolysin fortherapeutic purposes, the fundamental role of the enzyme is needed. In this study, an identical endolysin was identified from two C. difficile phages, ΦHN16-1 and ΦHN50, designated as CD16/50. The sequence analysis revealed a modular architecture of an N-terminal enzymatically active domain (EAD) and a C-terminal cell-wall binding domain(CBD). In vitro characterization confirmed that the EAD possessed bacteriolytic activitywhile the CBD bound bacterial cell-wall polysaccharide. The EAD alone exhibited lyticactivity faster than the full-length, suggesting a CBD-independent activity. The studyalso showed that the CBD formed a homodimer essential for interaction with cell-wallpolymer. Interestingly, the hidden Markov model analysis suggested that the CBD islikely derived from the CWB2 motif of C. difficile cell-wall proteins but exhibits ahigher binding affinity to bacterial cell-wall polysaccharides. Finally, endolysindiffusion and sequential cytolytic assays suggested that CBD is required for theendolysin to be trapped into post-lytic bacterial remnants, implying its physiologicalroles in limiting enzyme diffusion, preserving neighboring host cells, and allowing thephage progeny to initiate new rounds of infection. Collectively, this study showed the role of CBD in endolysin regulation, which may provide an insight into designing potentendolysins against C. difficile.