Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Unusual 1-3 peptidoglycan cross-links in Acetobacteraceae 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 four 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 which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that 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 bioinformatics approach, we identified a G. oxydans mutant from a transposon library 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.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

Molecular basis for substrate recognition and septum cleavage by AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis.

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved ß-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA-substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b.

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

Decoration of the enterococcal polysaccharide antigen EPA is essential for virulence, cell surface charge and interaction with effectors of the innate immune system.

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 a complex network of transcriptional regulators and 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.

Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases.

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved ß-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.

Bacterial size matters: Multiple mechanisms controlling septum cleavage and diplococcus formation are critical for the virulence of the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen frequently isolated in clinical settings. This organism is intrinsically resistant to several clinically relevant antibiotics and can transfer resistance to other pathogens. Although E. faecalis has emerged as a major nosocomial pathogen, the mechanisms underlying the virulence of this organism remain elusive. We studied the regulation of daughter cell separation during growth and explored the impact of this process on pathogenesis. We demonstrate that the activity of the AtlA peptidoglycan hydrolase, an enzyme dedicated to septum cleavage, is controlled by several mechanisms, including glycosylation and recognition of the peptidoglycan substrate. We show that the long cell chains of E. faecalis mutants are more susceptible to phagocytosis and are no longer able to cause lethality in the zebrafish model of infection. Altogether, this work indicates that control of cell separation during division underpins the pathogenesis of E. faecalis infections and represents a novel enterococcal virulence factor. We propose that inhibition of septum cleavage during division represents an attractive therapeutic strategy to control infections.

RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum.

BACKGROUND: The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq). RESULTS: We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 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. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (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 were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745. CONCLUSIONS: Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.

Towards an automated analysis of bacterial peptidoglycan structure.

Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3-3 and 4-3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3-3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported. Graphical Abstract The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry.

Prevalence and gene characteristics of antibodies with cofactor-induced HIV-1 specificity.

The healthy immune repertoire contains a fraction of antibodies that bind to various biologically relevant cofactors, including heme. Interaction of heme with some antibodies results in induction of new antigen binding specificities and acquisition of binding polyreactivity. In vivo, extracellular heme is released as a result of hemolysis or tissue damage; hence the post-translational acquisition of novel antigen specificities might play an important role in the diversification of the immunoglobulin repertoire and host defense. Here, we demonstrate that seronegative immune repertoires contain antibodies that gain reactivity to HIV-1 gp120 upon exposure to heme. Furthermore, a panel of human recombinant antibodies was cloned from different B cell subpopulations, and the prevalence of antibodies with cofactor-induced specificity for gp120 was determined. Our data reveal that upon exposure to heme, ∼24% of antibodies acquired binding specificity for divergent strains of HIV-1 gp120. Sequence analyses reveal that heme-sensitive antibodies do not differ in their repertoire of variable region genes and in most of the molecular features of their antigen-binding sites from antibodies that do not change their antigen binding specificity. However, antibodies with cofactor-induced gp120 specificity possess significantly lower numbers of somatic mutations in their variable region genes. This study contributes to the understanding of the significance of cofactor-binding antibodies in immunoglobulin repertoires and of the influence that the tissue microenvironment might have in shaping adaptive immune responses.

Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII.

Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria.

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N-Acetylmuramidase Activity.

UNLABELLED: Bacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novel Salmonella bacteriophage endolysin, Gp110, which comprises an uncharacterized domain of unknown function (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/µM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the ß-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents. IMPORTANCE: We report the functional and biochemical characterization of the Salmonella phage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 has N-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

A cryptic polyreactive antibody recognizes distinct clades of HIV-1 glycoprotein 120 by an identical binding mechanism.

Polyreactive antibodies play an important role for neutralization of human immunodeficiency virus (HIV). In addition to intrinsic polyreactive antibodies, the immune system of healthy individuals contains antibodies with cryptic polyreactivity. These antibodies acquire promiscuous antigen binding potential post-translationally, after exposure to various redox-active substances such as reactive oxygen species, iron ions, and heme. Here, we characterized the interaction of a prototypic human antibody that acquires binding potential to glycoprotein (gp) 120 after exposure to heme. The kinetic and thermodynamic analyses of interaction of the polyreactive antibody with distinct clades of gp120 demonstrated that the antigen-binding promiscuity of the antibody compensates for the molecular heterogeneity of the target antigen. Thus, the polyreactive antibody recognized divergent gp120 clades with similar values of the binding kinetics and quantitatively identical changes in the activation thermodynamic parameters. Moreover, this antibody utilized the same type of noncovalent forces for formation of complexes with gp120. In contrast, HIV-1-neutralizing antibodies isolated from HIV-1-infected individuals, F425 B4a1 and b12, demonstrated different binding behavior upon interaction with distinct variants of gp120. This study contributes to a better understanding of the physiological role and binding mechanism of antibodies with cryptic polyreactivity. Moreover, this study might be of relevance for understanding the basic aspects of HIV-1 interaction with human antibodies.

De-O-acetylation of peptidoglycan regulates glycan chain extension and affects in vivo survival of Neisseria meningitidis.

Peptidoglycan O-acetylation is a modification found in many bacteria. In Gram-positive pathogens, it contributes to virulence by conferring resistance to host lysozyme. However, in Gram-negative pathogens, its contribution to physiology and virulence is unknown. We examined the contribution of patA, patB and ape1 to peptidoglycan O-acetylation in the major human pathogen Neisseria meningitidis (Nm). Using genetic expression of all possible combinations of the three genes in Escherichia coli and Nm, we confirmed that PatA and PatB were required for PG O-acetylation, while ApeI removed the O-acetyl group. ApeI was active on all O-acetylated muropeptides produced by PatA and PatB during heterologous expression in E. coli and was also active on several PG structures in vitro. Interestingly, in Nm, ApeI was found to preferentially de-O-acetylate muropeptides with tripeptide stems (GM3), suggesting that its activity is highly regulated. Accordingly, de-O-acetylation of GM3 regulated glycan chain elongation and cell size. Additionally, the virulence of Nm lacking ApeI was drastically reduced suggesting that regulation of glycan chain length by O-acetylation contributes to bacterial fitness in the host. Altogether, our results suggest that ApeI represents an attractive target for new drug development.

A diverse set of Enterococcus-infecting phage provides insight into phage host-range determinants.

Enterococci are robust Gram-positive bacteria that pose a significant threat in healthcare settings due to antibiotic resistance, with vancomycin-resistant enterococci (VRE) most prominent. To tackle this issue, bacteriophages (bacterial viruses) can be exploited as they specifically and efficiently target bacteria. Here, we successfully isolated and characterised a set of novel phages: SHEF10, SHEF11, SHEF13, SHEF14, and SHEF16 which target E. faecalis (SHEF10,11,13), or E. faecium (SHEF13, SHEF14 & SHEF16) strains including a range of clinical and VRE isolates. Genomic analysis shows that all phages are strictly lytic and diverse in terms of genome size and content, quickly and effectively lysing strains at different multiplicity of infections. Detailed analysis of the broad host-range SHEF13 phage revealed the crucial role of the enterococcal polysaccharide antigen (EPA) variable region in its infection of E. faecalis V583. In parallel, the discovery of a carbohydrate-targeting domain (CBM22) found conserved within the three phage genomes indicates a role in cell surface interactions that may be important in phage-bacterial interactons. These findings advance our comprehension of phage-host interactions and pave the way for targeted therapeutic strategies against antibiotic-resistant enterococcal infections.

Zebrafish as a novel vertebrate model to dissect enterococcal pathogenesis.

Enterococcus faecalis is an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis. E. faecalis infections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing to E. faecalis virulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimic E. faecalis infections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first time E. faecalis pathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studying E. faecalis pathogenesis, enabling us to dissect the mechanism of enterococcal virulence.

A moonlighting role for LysM peptidoglycan binding domains underpins Enterococcus faecalis daughter cell separation.

Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.

The lysozyme-induced peptidoglycan N-acetylglucosamine deacetylase PgdA (EF1843) is required for Enterococcus faecalis virulence.

Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan O-acetylation, which inhibits the enzymatic activity of lysozyme, and partly by d-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss- and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.