Given their potential as specific and natural biocontrol agents, bacteriophages and their associated proteins have become the focus of renewed attention over the last decade. The aim of this study was to use a comparative modelling approach to generate a predicted 3D structure for LysB; a 332 amino acid lipolytic enzyme encoded by the mycobacteriophage Ardmore. The GXSXG pentapeptide, characteristic of lipolytic enzymes, was located at amino acid position 166-170. The three absolutely conserved residues among mycobacteriophage LysB proteins were also identified in Ardmore LysB as Ser-168, Gly-203 and Pro-205. CATH analysis of Ardmore LysB revealed a mainly Beta classification, Beta Barrel architecture and a topology similar to maltoporin. This is unlike the α/β hydrolase structure reported for the D29 LysB protein and, in fact appears in only 3 other sequenced LysB homologues to date. A search for conserved motifs within the amino acid sequence of LysB revealed the presence of both a cutinase motif and a PE-PPE motif. This study presents an in silico 3D predictive model of Ardmore lysin and confirms the high diversity of mycobacteriophages LysB proteins both at the sequence (2D) and structural (predicted 3D) levels.

Staphylococcus aureus is a major cause of infection in humans and animals, causing a wide variety of diseases, from local inflammations to fatal sepsis. The bacterium is commonly multi-drug resistant and thus many front-line antibiotics have been rendered ineffective for treating such infections. Research on murein/peptidoglycan hydrolases, derived from bacterial viruses (bacteriophages), has demonstrated that such proteins are attractive candidates for development as novel antibacterial agents for combatting Gram-positive pathogens. Here we review the research produced to-date on the bacteriophage-derived CHAPK murein peptidase. Initially, we sequenced and annotated the genome of anti-staphylococcal bacteriophage K and cloned the gene for the bacteriophage endolysin, a murein hydrolase which plays a role in cell killing during the bacteriophage life cycle. An highly active domain of the enzyme, a cysteine, histidine-dependent amido hydrolase/peptidase (CHAPK), was cloned, overexpressed in E. coli and purified. This CHAPK enzyme was demonstrated to rapidly lyse several strains of methicillin resistant S. aureus and both disrupted and prevented the formation of a staphylococcal biofilm. The staphylolytic activity of the peptidase was demonstrated in vivo using a mouse model, without adverse effects on the animals. The crystal structure of the enzyme was elucidated, revealing a calcium ion close to the active site. Site-directed mutagenesis indicated that this calcium ion is involved in the catalytic mechanism of the enzyme. The crystal structure of this enzyme is a valuable source of information for efficient engineering of this and similar CHAP-domain-containing proteins. Overall, the data collected to date on CHAPK has demonstrated its strong potential as a novel therapeutic candidate for treatment of staphylococcal infections and has provided us with insight into the fundamental enzymatic mechanisms of CHAP domain-containing peptidoglycan hydrolases.

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Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne’s disease in ruminants. As an infectious disease that causes reduced milk yields, effects fertility and, eventually, the loss of the animal, it is a huge financial burden for associated industries. Efforts to control MAP infection and Johne’s disease are complicated due to difficulties of diagnosis in the early stages of infection and challenges relating to the specificity and sensitivity of current testing methods. The methods that are available contribute to widely used test and cull strategies, vaccination programmes also in place in some countries. Next generation sequencing technologies have opened up new avenues for the discovery of novel biomarkers for disease prediction within MAP genomes and within ruminant microbiomes. Controlling Johne’s disease in herds can lead to improved animal health and welfare, in turn leading to increased productivity. With current climate change bills, such as the European Green Deal, targeting livestock production systems for more sustainable practices, managing animal health is now more important than ever before. This review provides an overview of the current knowledge on genomics and detection of MAP as it pertains to Johne’s disease.