Biology subjects

MESNAGE, S.

Publications and source records attributed to MESNAGE, S..

4 recordsLinked to original sources

bioRxiv2026

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.

microbiology↗

bioRxiv2026

Structural Basis for Dual Peptidoglycan Hydrolysis by an E. faecium Minhovirus Tail Spike Lysin

Bacteriophages rely on breaching the bacterial cell wall as the first step of infection. We characterise ORF11, a putative tail-spike lysin from the 19 kbp Minhovirus SHEF14, a podovirus infecting Enterococcus faecium. Bioinformatic analyses indicate that ORF11 comprises four domains: a predicted glycosyl hydrolase (D1) a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, D4), separated by a helical linker (D2) and a CHAP-like domain (D3). This modular organisation is conserved among Enterococcus minhoviruses but differs markedly from analogous proteins in Copernicusvirus phage and related staphylococcal phage. ORF11 2.1 [A] crystal structure reveals an unusual dimeric assembly. The predicted glycosyl hydrolase and CHAP peptidase domains occupy opposite ends of the protein, bridged by the two other domains positioned at the dimer interface. Biochemical assays using recombinant ORF11 and LC-MS/MS confirmed dual peptidoglycan-degrading activity. ORF11 functions as both an N-acetylglucosaminidase and a D, D-endopeptidase, cleaving the bond between the D-alanine in position-4 and the D-aspartate residue at the end of the side chain. Together, these results provide the first structural description of a podovirus tail-spike lysin and demonstrate its bifunctional enzymatic activity. This dual action likely facilitates initial surface recognition and localised peptidoglycan degradation during infection of E. faecium, offering new insights into how minimal-genome phage target this clinically significant antimicrobial-resistant pathogen.

biochemistry↗

bioRxiv2024

Exploring the role of E. faecalis Enterococcal Polysaccharide Antigen (EPA) and lipoproteins in evasion of phagocytosis

Enterococcus faecalis is an opportunistic pathogen frequently causing nosocomial infections. The virulence of this organism is underpinned by its capacity to evade phagocytosis, allowing dissemination in the host. Immune evasion requires a surface polysaccharide produced by all enterococci, known as the Enterococcal Polysaccharide Antigen (EPA). EPA consists of a cell wall-anchored rhamnose backbone substituted by strain-specific polysaccharides called "decorations", essential for the biological activity of this polymer. However, the structural determinants required for innate immune evasion remain unknown, partly due to a lack of suitable validated assays. Here, we describe a quantitative, in vitro assay to investigate how EPA decorations alter phagocytosis. Using the E. faecalis model strain OG1RF, we demonstrate that a mutant with a deletion of the locus encoding EPA decorations can be used as a platform strain to express heterologous decorations, thereby providing an experimental system to investigate the inhibition of phagocytosis by strain-specific decorations. We show that the aggregation of cells lacking decorations is increasing phagocytosis and that this process does not involve the recognition of lipoproteins by macrophages. Collectively, our work provides novel insights into innate immune evasion by enterococci and paves the way for further studies to explore the structure/function relationship of EPA decorations.

microbiology↗

bioRxiv2023

Unusual 1-3 peptidoglycan cross-links in Acetobacteriaceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position 4 of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software which can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a Sudoku transposon library, we identified a G. oxydans mutant with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

microbiology↗