Unlocking prophage potential:In silicoand experimental analysis of a novelMycobacterium fortuitumLysinB containing a peptidoglycan-binding domain

Abstract

Endolysins are highly evolved bacteriophage-encoded lytic enzymes produced to damage the bacterial cell wall for phage progeny release. They offer promising potential as highly specific lytic proteins with a low chance of bacterial resistance. The diversity in lysin sequences and domain organization can be staggering. In silico analysis of bacteriophage and prophage genomes can help identify endolysins exhibiting unique features and high antibacterial activity, hence feeding the pipeline of narrow-spectrum protein antibiotics. Mycobacteriophage lysis cassettes mostly have two lytic enzymes, LysinA and LysinB. The enzyme LysinA targets peptidoglycan in the cell wall and possesses a modular architecture. LysinB typically contains a single domain and acts upon the mycolyl ester linkages in mycolyl-arabinogalactan-peptidoglycan (Payne et al., 2010). This study aimed to find novel LysinBs against Mycobacterium fortuitum. After a detailed in silico characterization of lysis cassettes from three M. fortuitum prophages, we chose to work on a LysinB (hereafter described as LysinB_MF) found in an incomplete prophage (phiE1336, 9.4 kb in strain E1336). LysinB_MF showed low sequence similarity with any other endolysins in the database and formed a separate clade on phylogenetic analysis. LysinB_MFs structure, extracted from the AlphaFold Protein Structure Database, demonstrated a modular architecture with two structurally distinct domains: a peptidoglycan-binding domain (PGBD) at the N-terminal and the characteristic alpha/beta hydrolase domain connected via a linker peptide. We found the alpha/beta hydrolase domain, which is the enzyme-active domain (EAD), contains the conserved Ser-Asp-His catalytic triad with a tunnel-like topology and forms intermolecular hydrogen bonds. The PGBD shows structural similarity to the cell-wall binding domain of an amidase from Clostridium acetobutylicum, hinting at its acquisition due to domain mobility. Our in silico electrostatic potential analysis suggested that PGBD might be essential to the enzyme activity. This was experimentally validated by generating a truncated version of the enzyme, which demonstrated about six-fold decreased activity compared to its native form. The antimycobacterial activity of this enzyme was also compromised in its absence. Based on our analysis, PGBD emerged as an integral constituent of enzymes with diverse functional properties and is predicted to be a conserved cross-kingdom. Overall, this study highlights the importance of mining mycobacterial prophages as a novel endolysin source. It also provides unique insights into the diverse architecture of mycobacteriophage-encoded endolysins and the importance of functional domains for their catalytic activities.