Biology subjects

Mesnage, S.

Publications and source records attributed to Mesnage, S..

8 recordsLinked to original sources

bioRxiv2026

Rhizobial enzyme reveals pH-driven catalytic switching and ʟ-amino acid incorporation by ʟ,-transpeptidases

Nearly all bacteria are surrounded by a mesh-like macromolecule called peptidoglycan that gives them their shape and helps them resist turgor pressure. To grow and maintain their peptidoglycan, bacteria produce a wide range of enzymes, including the relatively understudied ,[x1D05]-transpeptidase (LDT) family. LDTs can catalyse several different reactions and vary widely in copy number: some bacteria have none, whilst others have more than twenty. To better understand why some bacteria have so many LDTs, we examined 18 putative ones from Rhizobium johnstonii, a nitrogen-fixing, symbiotic bacterium. Heterologous expression revealed several highly active enzymes, one of which, LdtRj8, we further characterized in detail. In vitro assays showed that LdtRj8 was capable of ,[x1D05]-transpeptidation, carboxypeptidation, substitution, and endopeptidation, but that its preferred activity differed at different pHs. LdtRj8 particularly excelled at ,[x1D05]-substitution, utilizing all of the tested [x1D05]-amino acids, and, surprisingly, most of the -amino acids as well. LdtRj8's pH-modulated activity could help R. johnstonii respond to acidic conditions encountered throughout the rhizobium-legume symbiosis, and its -amino acid substitution activity, which we show to be a more general property of LDTs, may regulate ,[x1D05]-transpeptidation and explain the existence of isomeric muropeptides often reported in the literature.

biochemistry↗

bioRxiv2024

Enterococcal cell wall remodelling underpins pathogenesis via the release of the Enteroccocal Polysaccharide Antigen (EPA)

Enterococci are opportunistic pathogens displaying a characteristic ovoid shape, typically forming pairs of cells (diplococci) and short chains. Control of cell chain length in Enterococcus faecalis relies on the activity of the major N-acetylglucosaminidase AtlA. The formation of short chains and diplococci is critical during pathogenesis for dissemination in the host and to limit recognition by innate immune effectors such as complement molecules and phagocytes. Here, we identify AtlE, an N-acetylmuramidase that contributes to septum cleavage during stationary phase in the absence of AtlA. AtlE is encoded by the locus required to produce the decoration subunits of the Enterococcal Polysaccharide Antigen (EPA), which mediate evasion of phagocytosis. We show that peptidoglycan hydrolysis by AtlE is essential for pathogenesis and demonstrate that soluble cell wall fragments containing EPA decorations increase the virulence of E. faecalis, suggesting that EPA plays a role as a decoy molecule to evade host defences. This research sheds light on the complex interplay between bacterial cell division, cell wall remodelling, and the host immune system, providing valuable insights into a novel mechanism underlying the virulence of E. faecalis. Author summaryThe major component of the bacterial cell envelope (peptidoglycan) undergoes partial hydrolysis during growth. This process, referred to as remodelling, is required for the incorporation of novel peptidoglycan building blocks, and cell separation at the end of division. In Enterococcus faecalis, only one ubiquitous peptidoglycan hydrolase, named AtlA, has been described so far. AtlA plays a prominent role in septum cleavage and is responsible for the characteristic formation of diplococci and short cell chains. The minimization of cell chain length by AtlA is critical for innate immune evasion and underpins pathogenesis. Here, we identify another ubiquitous peptidoglycan hydrolase named AtlE encoded by the Enterococcal Polysaccharide Antigen (EPA) biosynthetic locus. We show that AtlE displays N-acetylmuramidase activity and requires strain-specific EPA decorations to be active. Whilst AtlE only plays a marginal role in septum cleavage during growth, AtlE is essential for virulence in the zebrafish model of infection. We demonstrate that AtlE activity contributes to release cell wall fragments and promotes phagocyte evasion, indicating that EPA plays a role as a decoy molecule produced by enterococci to counteract host immune defenses.

microbiology↗

bioRxiv2024

The Enterococcal Polysaccharide Antigen: from structure to biosynthesis and function

L-Rhamnose-containing polysaccharides are produced by Streptococci and Enterococci. They define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The Enterococcal Polysaccharide Antigen produced by the opportunistic pathogen Enterococcus faecalis plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer for E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, we elucidate the structure of the intact EPA produced by E. faecalis OG1RF. We report the structure of the linkage unit, revealing an unprecedented complexity of the rhamnose backbone and decorations. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA for the rational design of therapeutic strategies against a group of important pathogens.

microbiology↗

bioRxiv2021

Human serum triggers antibiotic tolerance in Staphylococcus aureus

Staphylococcus aureus is a frequent cause of bloodstream infections. Treatment can be challenging, even when isolates appear to be drug susceptible, with high rates of persistent and relapsing infection. This is particularly the case with infections caused by methicillin resistant S. aureus (MRSA) strains, which are resistant to frontline antibiotics. To understand how the host environment influences treatment outcomes in MRSA infections, we studied the impact of human serum on staphylococcal susceptibility to daptomycin, an antibiotic of last resort. This revealed that serum triggered a very high degree of tolerance to daptomycin, as well as several other classes of antibiotics and antimicrobial peptides, including gentamicin, nitrofurantoin, vancomycin, nisin and gramicidin. Serum-induced daptomycin tolerance was due to two independent mechanisms. Firstly, the host defence peptide LL-37 present in serum induced tolerance by triggering the staphylococcal GraRS two component system. This led to increased cell wall accumulation that reduced access of daptomycin to its membrane target. Secondly, GraRS-independent changes to the membrane resulted in increased cardiolipin abundance that also contributed to daptomycin tolerance. When both mechanisms were blocked, serum exposed S. aureus cells were as susceptible to daptomycin as bacteria growing in laboratory media. These data demonstrate that host factors can significantly modulate antibiotic susceptibility via diverse mechanisms, which may in turn contribute to treatment failure. The inhibition of serum-induced cell wall accumulation by fosfomycin reduced tolerance, suggesting that this antibiotic may form a useful combination therapy with daptomycin.

microbiology↗

bioRxiv2021

PGfinder, a novel analysis pipeline for the consistent, reproducible and high- resolution structural analysis of bacterial peptidoglycans

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan, the essential and major component of bacterial cell envelopes. It is comprised of glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGfinder for the analysis of peptidoglycan structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial peptidoglycans. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

microbiology↗

bioRxiv2020

A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

Type VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella SPI-6 T6SS antibacterial effector called Tae5STM (type VI amidase effector 5). Tae5STM is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STM and the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STM displays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and D-alanine4. Phylogenetic analysis showed that Tae5STM homologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among -, {beta}- and {gamma}-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.

microbiology↗

bioRxiv2018

Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and sensitivity to effector molecules of innate immunity

Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.

microbiology↗

bioRxiv2017

Fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum

Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. Here, we identify genes that contribute to growth of E. faecium in human serum. We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, to completion and then compared its transcriptome during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. A high-throughput transposon sequencing (Tn-seq) approach was used to identify conditionally essential genes in E. faecium E745 during growth in serum. Genes involved in de novo nucleotide biosysnthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants also exhibited significantly attenuated virulence in an intravenous zebrafish infection model. We conclude that genes involved in carbohydrate and nucleotide metabolism of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism.

microbiology↗