In Vitro Characterization of PHICD111_20024_EAD as an Engineered Endolysin against Clostridioides difficile
Abstract
OBJECTIVES
Clostridioides difficile is a gram-positive, spore-forming obligate anaerobe that can cause symptoms such as diarrhea and abdominal cramping and lead to conditions including pseudomembranous colitis. The mainstay treatment of C. difficile infection (CDI) is antibiotics; however, antibiotics-induced gut microbiota dysbiosis poses a potential risk for refractory CDI and increased CDI recurrences. Recently, there has been growing interest in bacteriophages and their lytic enzymes as alternatives to antibiotics. We aimed to develop an engineered endolysin to specifically inactivate C. difficile.
METHODS
Endolysins are bacteriolytic enzymes that facilitate the release of phage progeny during the final stage of infection. We previously demonstrated that CD27L_EAD, an engineered endolysin, inactivated C. difficile in a co-culture model of human gut microbiota. This study modified the ΦCD111 endolysin to PHICD111_20024_EAD by cleaving the cell wall-binding domain and characterized its catalytic activity in comparison with CD27L_EAD.
RESULTS
PHICD111_20024_EAD shows high sequence similarity with CD27L_EAD. However, PHICD111_20024_EAD exerted superior bacteriolytic activity compared to CD27L_EAD under high salt concentrations. PHICD111_20024_EAD and CD27L_EAD displayed similar overall structures, consisting of five α-helices and six β-strands, and were superimposed with a zinc ion at the active site, suggesting that both enzymes are zinc-dependent N-acetylmuramoyl-L-alanine…
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