Multiple Campylobacter jejuni proteins affecting the peptidoglycan structure and the degree of helical cell curvature.

Campylobacter jejuni is a Gram-negative helical bacterium. Its helical morphology, maintained by the peptidoglycan (PG) layer, plays a key role in its transmission in the environment, colonization, and pathogenic properties. The previously characterized PG hydrolases Pgp1 and Pgp2 are important for generating C. jejuni helical morphology, with deletion mutants being rod-shaped and showing alterations in their PG muropeptide profiles in comparison to the wild type. Homology searches and bioinformatics were used to identify additional gene products involved in C. jejuni morphogenesis: the putative bactofilin 1104 and the M23 peptidase domain-containing proteins 0166, 1105, and 1228. Deletions in the corresponding genes resulted in varying curved rod morphologies with changes in their PG muropeptide profiles. All changes in the mutants complemented except 1104. Overexpression of 1104 and 1105 also resulted in changes in the morphology and in the muropeptide profiles, suggesting that the dose of these two gene products influences these characteristics. The related helical ε-Proteobacterium Helicobacter pylori has characterized homologs of C. jejuni 1104, 1105, and 1228 proteins, yet deletion of the homologous genes in H. pylori had differing effects on H. pylori PG muropeptide profiles and/or morphology compared to the C. jejuni deletion mutants. It is therefore apparent that even related organisms with similar morphologies and homologous proteins can have diverse PG biosynthetic pathways, highlighting the importance of studying PG biosynthesis in related organisms.

Two New M23 Peptidoglycan Hydrolases With Distinct Net Charge.

Bacterial peptidoglycan hydrolases play an essential role in cell wall metabolism during bacterial growth, division, and elongation (autolysins) or in the elimination of closely related species from the same ecological niche (bacteriocins). Most studies concerning the peptidoglycan hydrolases present in Gram-positive bacteria have focused on clinically relevant Staphylococcus aureus or the model organism Bacillus subtilis, while knowledge relating to other species remains limited. Here, we report two new peptidoglycan hydrolases from the M23 family of metallopeptidases derived from the same staphylococcal species, Staphylococcus pettenkoferi. They share modular architecture, significant sequence identity (60%), catalytic and binding residue conservation, and similar modes of activation, but differ in gene distribution, putative biological role, and, strikingly, in their isoelectric points (pIs). One of the peptides has a high pI, similar to that reported for all M23 peptidases evaluated to date, whereas the other displays a low pI, a unique feature among M23 peptidases. Consequently, we named them SpM23_B (Staphylococcus pettenkoferi M23 "Basic") and SpM23_A (Staphylococcus pettenkoferi M23 "Acidic"). Using genetic and biochemical approaches, we have characterized these two novel lytic enzymes, both in vitro and in their physiological context. Our study presents a detailed characterization of two novel and clearly distinct peptidoglycan hydrolases to understand their role in bacterial physiology.

Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases.

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved ß-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.

Solution structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain of Zoocin A.

Zoocin A is a Zn-metallopeptidase secreted by Streptococcus zooepidemicus strain 4881. Its catalytic domain is responsible for cleaving the D-alanyl-L-alanine peptide bond in streptococcal peptidoglycan. The solution NMR structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain (rCAT C74A) has been determined. With a previous structure determination for the recombinant target recognition domain (rTRD), this completes the 3D structure of zoocin A. While the structure of rCAT C74A resembles those of the catalytic domains of lysostaphin and LytM, the substrate binding groove is wider and no tyrosine residue was observed in the active site. Proteins 2016; 85:177-181. © 2016 Wiley Periodicals, Inc.

Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent.

Despite intense efforts by the medical and pharmaceutical communities, Staphylococcus aureus continues to be a pervasive pathogen that causes a myriad of diseases and a high level of morbidity and mortality among infected patients. Thus, discovering or designing novel therapeutics able to kill both drug-resistant and drug-sensitive S. aureus remains a top priority. Bacteriolytic enzymes, mostly from phage, have shown great promise in preclinical studies, but little consideration has been given to cis-acting autolytic enzymes derived from the pathogen itself. Here, we use the S. aureus autolysin LytM as a proof of principal to demonstrate the antibacterial potential of endogenous peptidoglycan-degrading enzymes. While native LytM is only marginally bactericidal, fusion of LytM to the lysostaphin cell wall binding domain enhances its anti-staphylococcal activity approximately 540-fold, placing it on par with many phage lysins currently in preclinical development. The potential to therapeutically co-opt a pathogen's endogenous peptidoglycan recycling machinery opens the door to a previously untapped reservoir of antibacterial drug candidates.