In vitro characterization of PHICD111_20024_EAD as an engineered endolysin against Clostridioides difficile.

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.