Structure and metabolism of peptidoglycan and molecular requirements allowing its detection by the Drosophila innate immune system

Peptidoglycan (murein) is a major essential and specific constituent of the bacterial cell wall. Its main function is to protect cells against the internal osmotic pressure and to maintain the characteristic cell shape. It also serves as a platform for the anchoring of specific proteins and other cell wall components. This giant macromolecule is composed of long glycan chains cross-linked by short peptides. Any alteration of the disaccharide-peptide basic unit results in a global change of peptidoglycan structure and properties. Such global variations are encountered in nature as conserved variations along phyletic lines but have sometimes been acquired as a result of mutations or as a mechanism of resistance against cell-wall targeted antibiotics. During bacterial cell growth and division, the peptidoglycan mesh is constantly broken down by a set of highly specific hydrolases in a maturation process allowing insertion of newly synthesized units in the pre-existing polymerized material. Depending on the bacterial species considered, degradation fragments are either released in the growth medium or efficiently re-utilized for synthesis of new murein in a sequence of events termed the recycling pathway. Peptidoglycan is one of the main pathogen-associated molecular patterns recognized by the host innate immune system. Variations of the structure and metabolism of this cell wall component have been exploited by host defense mechanisms for detection/identification of invading bacterial species. Modification of the peptidoglycan structure could also represent a mechanism allowing bacteria to escape these host defense systems

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa.

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans

Periplasmic Phosphorylation of Lipid a Is Linked to the Synthesis of Undecaprenyl Phosphate

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. 32P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo2-[4′- 32P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan

The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP11/52067-6; 2017/12436-9; 2013/02451-0FRISBI [ANR-10-INSB-05-02]; GRAL within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-LABX-49-01]; Rhone-Alpes RegionRegion Auvergne-Rhone-Alpes; Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale; fonds FEDER; Centre National de la Recherche Scientifique (CNRS)Centre National de la Recherche Scientifique (CNRS); Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA)French Atomic Energy Commission; University of Grenoble Alpes; EMBL; GIS-Infrastructures en Biologie Sante et Agronomie (IBISA); Laboratoire International Associe BACWALL (CNRS); FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [11/52067-6, 2017/12436-9]; Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-13-BSV8-0015-01]; ANRFrench National Research Agency (ANR); Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale [FDT20160435484]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/02451-0

Specificity determinants for lysine incorporation in staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex

Background: MurE controls stereo chemical incorporation of Lysine or diaminopimelate into peptidoglycan stem peptides Results: The structure of S.aureus MurE reveals an unexpected lack of specificity for Lysine within the active site. Conclusion: Incorporation of Lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity. Significance: This study provides new perspectives in targeting Gram-positive peptidoglycan assembly for antimicrobial discovery

Role of AmiA in the Morphological Transition of Helicobacter pylori and in Immune Escape

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-β(1,4)-N-acetylmuramyl-L-Ala–D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-κB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection

Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in <em>Escherichia coli</em>

International audienceThe target of β-lactam antibiotics is the D,D-transpeptidase activity of penicillin-binding proteins (PBPs) for synthesis of 4→3 cross-links in the peptidoglycan of bacterial cell walls. Unusual 3→3 cross-links formed by L,D-transpeptidases were first detected in Escherichia coli more than four decades ago, however no phenotype has previously been associated with their synthesis. Here we show that production of the L,D-transpeptidase YcbB in combination with elevated synthesis of the (p)ppGpp alarmone by RelA lead to full bypass of the D,D-transpeptidase activity of PBPs and to broad-spectrum β-lactam resistance. Production of YcbB was therefore sufficient to switch the role of (p)ppGpp from antibiotic tolerance to high-level β-lactam resistance. This observation identifies a new mode of peptidoglycan polymerization in E. coli that relies on an unexpectedly small number of enzyme activities comprising the glycosyltransferase activity of class A PBP1b and the D,D-carboxypeptidase activity of DacA in addition to the L,D-transpeptidase activity of YcbB

The Drosophila immune system detects bacteria through specific peptidoglycan recognition

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan

The essential peptidoglycan glycosyltransferase MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli

In Escherichia coli many enzymes including MurG are directly involved in the synthesis and assembly of peptidoglycan. MurG is an essential glycosyltransferase catalysing the last intracellular step of peptidoglycan synthesis. To elucidate its role during elongation and division events, localization of MurG using immunofluorescence microscopy was performed. MurG exhibited a random distribution in the cell envelope with a relatively higher intensity at the division site. This mid-cell localization was dependent on the presence of a mature divisome. Its localization in the lateral cell wall appeared to require the presence of MreCD. This could be indicative of a potential interaction between MurG and other proteins. Investigating this by immunoprecipitation revealed the association of MurG with MreB and MraY in the same protein complex. In view of this, the loss of rod shape of ΔmreBCD strain could be ascribed to the loss of MurG membrane localization. Consequently, this could prevent the localized supply of the lipid II precursor to the peptidoglycan synthesizing machinery involved in cell elongation. It is postulated that the involvement of MurG in the peptidoglycan synthesis concurs with two complexes, one implicated in cell elongation and the other in division. A model representing the first complex is proposed

Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell