Characterization of mycophage endolysin cell wall binding domains targeting Mycobacterium bovis peptidoglycan

• Published in: Biochemical and biophysical research communications Vol. 681; pp. 291 - 297
• Main Authors: Itterbeek, Annabel, Possemiers, Amber, Colak, Yunus, Bäcker, Leonard E., Aertsen, Abram, Lavigne, Rob, Paeshuyse, Jan
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 12.11.2023
• Subjects: PGRP; bTB; CBD; VTS; LB; BCG; His-tag; IPTG; GlcNAc; MurNGlyc; MurNAc; Ni-NTA
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2023.09.027

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis, responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics. •Previously characterized pfam 01471 domain was annotated in mycophage endolysins.•Mycophage endolysins contain new, uncharacterized cell-wall binding domains.•Cell-wall binding domains fused to GFP fluorescently labelled M. bovis BCG in milk.