Data mining reveals the diversity of prophage endolysins targeting pathogenic enterococci

Antimicrobial resistance (AMR) poses a critical global health threat, with enterococci among the leading contributors due to their intrinsic and acquired resistance to antibiotics. Clinically relevant species, including Enterococcus faecalis and Enterococcus faecium, as well as the emerging poultry pathogen Enterococcus cecorum, highlight the need for alternative therapeutics across human and agricultural settings. Bacteriophages and their derived enzymes, particularly endolysins, offer promising antibacterial strategies but challenges such as phage resistance and limited lysin diversity hinder their application. In this study, we performed a large-scale analysis of prophage-encoded endolysins across these three enterococcal opportunistic pathogens, characterizing over 48,000 sequences. We identified 33 distinct domain architectures combining diverse catalytic and cell wall-binding domains, including novel putative cell wall binding domains. These findings expand the known diversity of enterococcal lysins and provide a comprehensive resource for the rational design of stable, recombinant "enzybiotics" to combat multidrug-resistant enterococcal infections. Data summaryAll genomes analysed in this work are available through Genbank. The data mining strategy was carried out open-access software available through GitHub as described in the Methods section. The raw output of the search and sequences obtained after each filtering step are provided in Supplementary Files 1 and 2. Modelling data related to figure 6 is provided in supplementary File 3. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/720912v1_fig6.gif" ALT="Figure 6"> View larger version (42K): org.highwire.dtl.DTLVardef@16ea8f5org.highwire.dtl.DTLVardef@16883aorg.highwire.dtl.DTLVardef@149d360org.highwire.dtl.DTLVardef@1d47ec1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO BSD is predicted to be a novel cell wall binding domain. A, Alignment of amino acid sequences encoding the BSD. B, Structural alignment of AlphaFold predictions. C, A box plot quantifying the ligand ipTM scores of 25 Efm and Efs muropeptide dockings for various domains / proteins, and the custom-calculated docking consistency scores for each of those 25 predictions. The results have been visually grouped into predicted binders and predicted non-binders by comparing ipTM and docking consistency scores to known binders (LysM, SH3) and random proteins (GFP, RNBR). D, Docking of Efs muropeptide (gold) into the binding pocket of BSD (blue), including any predicted hydrogen bonding. C_FIG Impact statementAntimicrobial resistant enterococci threaten therapeutic options in both medicine and agriculture. Yet, the therapeutic potential of bacteriophage-derived endolysins (enzybiotics) is limited by an incomplete understanding of their natural diversity. By analysing more than 48,000 prophage encoded lysins from E. faecalis, E. faecium, and E. cecorum, this study provides the most extensive characterization of enterococcal lysin architectures to date. The identification of 34 distinct domain organizations, including a previously unrecognized cell wall-binding domain, substantially broadens the known functional repertoire of these enzymes. This work fills a major knowledge gap and offers a foundational resource for engineering stable, targeted enzybiotics to combat multidrug resistant enterococcal infections.