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Updated: May 10, 2026
Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291
Published on: December 10, 2016
In vitro characterization of PHICD111_20024_EAD as an engineered endolysin against Clostridioides difficile
Jeongseok Park1, Siyoung Yoo1, Jieun An1
1Department of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea.
Objective:
Clostridioides difficile is a Gram-positive, spore-forming obligate anaerobe that can cause symptoms such as diarrhoea 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 amidases. However, their binding affinities for zinc ions differed, and excess zinc ions inhibited the catalytic activity of PHICD111_20024_EAD.
Conclusions:
For feasible applications of endolysins in dynamic gut environments, PHICD111_20024_EAD, which is tolerant to high osmolarity and can be modulated using zinc concentrations, provides advantageous attributes for developing therapeutic agents against CDI.
Insights
Engineered endolysins offer a promising alternative to antibiotics for treating Clostridioides difficile infections (CDI). PHICD111_20024_EAD shows enhanced activity and adaptability for gut environments, aiding CDI therapeutic development.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Clostridioides difficile infection (CDI) is a significant health concern, often treated with antibiotics that can lead to dysbiosis and recurrence.
- Bacteriophages and their lytic enzymes are emerging as potential alternatives to conventional antibiotic therapies for CDI.
- Engineered endolysins present a targeted approach to combat bacterial infections like CDI.
Purpose of the Study:
- To develop and characterize an engineered endolysin, PHICD111_20024_EAD, for specific inactivation of Clostridioides difficile.
- To compare the catalytic activity and structural properties of PHICD111_20024_EAD with a previously studied endolysin, CD27L_EAD.
Main Methods:
- Modification of the ΦCD111 endolysin by cleaving the cell wall-binding domain to create PHICD111_20024_EAD.
- Comparative analysis of the bacteriolytic activity of PHICD111_20024_EAD and CD27L_EAD under varying salt concentrations.
- Structural characterization and assessment of zinc ion dependency and inhibition for both engineered endolysins.
Main Results:
- PHICD111_20024_EAD demonstrated superior bacteriolytic activity against C. difficile compared to CD27L_EAD, particularly under high salt conditions.
- Both endolysins share similar structural features, identified as zinc-dependent N-acetylmuramoyl-L-alanine amidases.
- Differences in zinc ion binding affinity were observed, with excess zinc inhibiting PHICD111_20024_EAD activity.
Conclusions:
- PHICD111_20024_EAD exhibits advantageous attributes for therapeutic applications against CDI due to its tolerance to high osmolarity.
- The activity of PHICD111_20024_EAD can be modulated by zinc concentrations, offering a controllable therapeutic approach.
- This engineered endolysin represents a promising candidate for developing novel anti-CDI agents suitable for dynamic gut environments.
