ABSTRACT Enterococci are opportunistic pathogens classified by the World Health Organization as high-priority microorganisms. They cause a broad spectrum of infections, and their intrinsic and acquired resistance to antimicrobials makes these infections particularly difficult to treat and eradicate. In Enterococcus faecalis , the most frequently isolated enterococcal pathogen in humans, antimicrobial resistance and innate immune evasion are largely driven by the Enterococcal Polysaccharide Antigen (EPA). This surface polymer underpins key virulence traits, including resistance to host defence mechanisms, reduced susceptibility to multiple classes of antimicrobials, and susceptibility to bacteriophage infection. EPA consists of a rhamnan backbone decorated with strain-specific substituents that are essential for its biological activity. Here, we show that epaB encodes the enzyme responsible for the first committed step in assembling the EPA rhamnan chain. Using NMR spectroscopy, we demonstrate that E. faecalis lacking epaB produces an EPA polymer composed solely of decorations directly anchored to the peptidoglycan, with no detectable rhamnan backbone. The absence of this rhamnan moiety profoundly alters cell wall architecture, as revealed by atomic force microscopy of the mutant cell walls. The epaB mutation also abolishes innate immune evasion and virulence in the zebrafish infection model, while conferring resistance to bacteriophages. Collectively, these findings demonstrate that both the rhamnan backbone and its decorations are required for EPA’s full biological activity, establishing the structural and functional interdependence of these two components. IMPORTANCE The Enterococcal Polysaccharide Antigen (EPA) is essential for normal growth and division, virulence, antimicrobial resistance, and phage infection in enterococci. This surface polymer comprises a structurally conserved rhamnan backbone substituted with strain-specific decorations. These variable decorations have been directly linked to the biological functions of EPA, whereas the rhamnan backbone has been proposed to serve primarily as a structural scaffold. Here, we use NMR spectroscopy to show that mutation of epaB , the gene responsible for the first biosynthetic step in rhamnan backbone formation, results in an EPA polymer composed exclusively of decorations anchored to the peptidoglycan. We reveal that the lack of rhamnan backbone is associated with a change in the cell wall architecture and abolishes virulence and infection by bacteriophages. Together, these findings demonstrate that assembly of the conserved rhamnan backbone is indispensable for EPA function and highlight this biosynthetic step as a promising target to combat enterococcal infections.

Bacterial exopolysaccharides (EPS) are carbohydrate polymers secreted into the environment. EPS produced by lactic acid bacteria have many valuable properties in the food and health sectors. In this study, we isolated spontaneous mutants of lactobacilli that overproduce EPS, using a selection method based on their slow sedimentation rate in a semi-liquid medium. In the mutants selected from several strains, we detected a missense mutation in epsD, which encodes a tyrosine kinase, or an insertion in epsC, which encodes its transmembrane modulator. Both genes were located within a gene cluster involved in Wzy-dependent polysaccharide biosynthesis. We then characterized selected Lacticaseibacillus rhamnosus mutants in detail to gain insights into the mechanisms involved in EPS overproduction. We demonstrated that the single mutation D94L in the EpsD catalytic site prevents EpsD autophosphorylation. The chemical structure of the overproduced EPS was established, and consists of heptasaccharide repeating units with pyruvate substituents. In the wild-type parental strain, a polysaccharide with an identical structure was found covalently bound to the cell wall (CW) and covering the bacterial surface. In conclusion, our results indicate that the switch from CW-bound polysaccharides to EPS released into the environment is associated with a defect in autophosphorylation of the EpsD tyrosine kinase.

Exopolysaccharides (EPS) were purified from two Lactiplantibacillus pentosus spontaneous EPS-overproducing mutants, derived from two strains originating from fermented vegetal products. Their chemical structure was established by methylation analysis, 2D NMR spectroscopy, and mass spectrometry. The first EPS isolated from strain VES7988 has an original structure made of O-acetylated sialyl-lactose repeating units. The second EPS isolated from strain VES8020 is an acidic polysaccharide made of linear nonasaccharide repeating units linked by phosphodiester bonds. The third EPS also isolated from VES8020 is a neutral polysaccharide made of branched pentasaccharide repeating units.

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.

Bacillus subtilis spores persist on food-processing equipment surfaces, contributing to the contamination of finished products and food loss. Their outermost layer, the crust, influences spore adhesion, notably through its glycans, whose structures remain unknown. Here, we report the identification and structural characterization of quinovosan, a novel homopolymeric polysaccharide composed of D-quinovose residues found in a non-covalently bound form within the crust. We showed that quinovosan consists of two-thirds α- and one-third β-D-quinovosyl residues. Of the nine quinovosyl residues forming the repeating unit of quinovosan, five were monosubstituted at O-3 and two of them were disubstituted at O-2 and O-3, and finally two were in non-reducing terminal positions. Our findings also indicate that quinovosan slightly contributes to spore adhesion and plays a key role in crust architecture. Its amphiphilic nature may promote interactions between hydrophilic molecules and hydrophobic proteins, thereby contributing to the supramolecular organization of the crust. Finally, we identified the yfnHGFED operon as responsible for quinovosan biosynthesis. Specifically, yfnH and yfnG are required for quinovose biosynthesis, while yfnF, yfnE, and yfnD likely participate in quinovosan assembly. Based on these findings, we propose a quinovosan biosynthetic pathway in B. subtilis.

The Gram-positive bacterium Oenococcus oeni is a major player in wine malolactic fermentation. In O. oeni, cell wall polysaccharides are considered putative receptors for bacteriophages, virus predators that lead to fermentation failures. In this study, we have developed an efficient stepwise extraction protocol to extract polysaccharides from the cell wall of O. oeni IOEBS277, which were analyzed by methylation, 1D, 2D-NMR spectroscopy, and MALDI-QIT-TOF mass spectrometry. The chemical structures of the two major purified polysaccharides were elucidated. The first one is a heteropolysaccharide with repeating units consisting of a branched hexasaccharide and one glycerol residue, linked by phosphodiester bonds. The second one consists of a →6)-β-Galf-(1→ galactofuranan chain partially substituted on the C-2 hydroxyl with β-Glcp. HR-MAS NMR analysis of intact O. oeni cells indicated that both polysaccharides are exposed to the bacterial surface.

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. ### Competing Interest Statement The authors have declared no competing interest.

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), 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 in E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, using an NMR approach, we elucidate the structure of EPA and propose a model for biosynthesis. We report the structure of the EPA-peptidoglycan linkage unit and reveal an unprecedented complexity of the EPA rhamnose backbone and decoration subunits. 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 and the rational design of therapeutic strategies against a group of important pathogens.

Chemosynthetic bacteria play an important role in supporting the ecosystem in deep-sea hydrothermal fields. Many deep-sea vent endemic animals (e.g. arthropods, gastropods, and bivalves) depend on specific microbial partners that they acquire in every generation, however, the mechanism by which these symbiotic microorganisms are acquired and maintained remains unknown. Glycans present in all three domains of life, and often serve critical functions in various biological interactions. Recent genome-based studies have suggested that deep-sea chemosynthetic bacteria frequently have the ability to produce unique glycans. In this study, we analyzed the glycan structure of the episymbiotic microbial biofilm associated with a deep-sea vent squat lobster, Shinkaia crosnieri. The hydrazinolysis-released glycan was labelled with 2-aminopyridine and then analyzed using RP-HPLC, CE-MS, and NMR. The purified tetrasaccharide had a molecular structure of 4,5-α-D-ene-glucuronic acid, 1,4-β-D-glucose, 1,4-α-D-methyl rhamnose, 1,3-D-galactose, which was similar to the repeating unit of gellan gum. This study represents the first structural analysis of glycans produced by symbiotic bacteria in deep-sea hydrothermal fields.

The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.

La plateforme vient d'acquerir en janvier 2020 une electrophorese capillaire avec plusieurs types de detection comme l'UV ou la fluorescence. Celle-ci sera disponible aux utilisateurs des que les…

Cet article de H. A. Currie et C. C. Perry de 2006 est interessant et permet de revisiter l'analyse des monosaccharides y compris des uronates (acide Galacturonique et Glucuronique) dans un…

All obligate intracellular pathogens or symbionts of eukaryotes lack glycogen metabolism. Most members of the Chlamydiales order are exceptions to this rule as they contain the classical GlgA-GlgC-dependent pathway of glycogen metabolism that relies on the ADP-Glucose substrate. We surveyed the diversity of Chlamydiales and found glycogen metabolism to be universally present with the important exception of Criblamydiaceae and Waddliaceae families that had been previously reported to lack an active pathway. However, we now find elements of the more recently described GlgE maltose-1-P-dependent pathway in several protist-infecting Chlamydiales. In the case of Waddliaceae and Criblamydiaceae , the substitution of the classical pathway by this recently proposed GlgE pathway was essentially complete as evidenced by the loss of both GlgA and GlgC. Biochemical analysis of recombinant proteins expressed from Waddlia chondrophila and Estrella lausannensis established that both enzymes do polymerize glycogen from trehalose through the production of maltose-1-P by TreS-Mak and its incorporation into glycogen’s outer chains by GlgE. Unlike Mycobacteriaceae where GlgE-dependent polymerization is produced from both bacterial ADP-Glc and trehalose, glycogen synthesis seems to be entirely dependent on host supplied UDP-Glc and Glucose-6-P or on host supplied trehalose and maltooligosaccharides. These results are discussed in the light of a possible effector nature of these enzymes, of the chlamydial host specificity and of a possible function of glycogen in extracellular survival and infectivity of the chlamydial elementary bodies. They underline that contrarily to all other obligate intracellular bacteria, glycogen metabolism is indeed central to chlamydial replication and maintenance.

Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA ( e nterococcal p olysaccharide a ntigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called “EPA decorations” essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.