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

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.

Genomic Sequence of Bacteriophage ATCC 8074-B1 and Activity of Its Endolysin and Engineered Variants against Clostridium sporogenes

“…Other studies reported the maintenance of endolysin activity upon removal of C-terminal domains; in many cases, this truncation led to a notable increase in activity (9,14,28,32), while in others, activity remained similar to or was lower than that of the full-length endolysin (10,21). In several cases, endolysin catalytic domains retained the same or a similar host range upon removal of the C-terminal domain (9,18,28,32), despite the fact that their peptidoglycan target is usually shared with a number of other species. The difference in lytic activities between CS74L and CS74L varied between the target strains of C. sporogenes; in the majority of cases, activity was broadly similar or slightly greater, although with some strains the full-length endolysin was clearly more effective.…”

“…CD27L, which targets C. difficile, is an N-acetylmuramoyl-Lalanine amidase endolysin whose truncated catalytic domain (CD27L ) shows an increased activity compared to the full endolysin (32). It shows some similarity to CS74L across the whole length of the proteins (41.7% consensus, 28.4% identity) but is not active against C. sporogenes cells.…”

“…In addition, some endolysin catalytic domains retain both their activity and a measure of specificity after removal of their C-terminal domains (9,28,32).…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…In addition, the deletion broadened the mutant enzyme's lytic range. 48 The mechanism for these outcomes was not determined. Likewise, the prophage LambdaSa2 endolysin has two EADs, an endopeptidase that is a highly catalytic and a poorly catalytic glycosidase.…”

“…31 Removal of terminal region amino acids from an endolysin can also improve an enzyme's bacteriolytic potency. 48 An N-terminal deletion of the Clostridium difficile phage endolysin CD27L improved bacteriolytic activity, despite only removing regions outside the predicted EAD and CBD. In addition, the deletion broadened the mutant enzyme's lytic range.…”