Structure-Based Modification of a Clostridium difficile-Targeting Endolysin Affects Activity and Host Range

Abstract

ABSTRACT Endolysin CD27L causes cell lysis of the pathogen Clostridium difficile , a major cause of nosocomial infection. We report a structural and functional analysis of the catalytic activity of CD27L against C. difficile and other bacterial strains. We show that truncation of the endolysin to the N-terminal domain, CD27L 1–179 , gave an increased lytic activity against cells of C. difficile , while the C-terminal region, CD27L 180–270 , failed to produce lysis. CD27L 1–179 also has increased activity against other bacterial species that are targeted by the full-length protein and in addition was able to lyse some CD27L-insensitive strains. However, CD27L 1–179 retained a measure of specificity, failing to lyse a wide range of bacteria. The use of green fluorescent protein (GFP)-labeled proteins demonstrated that both CD27L and CD27L 1–179 bound to C. difficile cell walls. The crystal structure of CD27L 1–179 confirms that the enzyme is a zinc-dependent N -acetylmuramoyl- l -alanine amidase. A structure-based sequence analysis allowed us to identify four catalytic residues, a proton relay cascade, and a substrate binding pocket. A BLAST search shows that the closest-related amidases almost exclusively target Clostridia . This implied that the catalytic domain alone contained features that target a specific bacterial species. To test this hypothesis, we modified Leu 98 to a Trp residue which is found in an endolysin from a bacteriophage of Listeria monocytogenes (PlyPSA). This mutation in CD27L resulted in an increased activity against selected serotypes of L. monocytogenes , demonstrating the potential to tune the species specificity of the catalytic domain of an endolysin.