Dominique Mengin-Lecreulx

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.

Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists

Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide‐binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro‐inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc‐L‐Ala‐γ‐D‐Glu‐meso‐diaminopimelic acid, MtriDAP) and NOD2 (MurNAc‐L‐Ala‐D‐isoGln, MDP; MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro‐inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro‐ and anti‐inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub‐active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll‐like receptor 4 to instruct the onset of adaptive immune responses.

Ampd, Essential for Both Beta-Lactamase Regulation and Cell Wall Recycling, Is a Novel Cytosolic N-Acetylmuramyl-L-Alanine Amidase

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of β‐lactamase expression. It is shown here that the AmpD protein is a novel N‐acetylmuramyl‐L‐alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptido‐glycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a‘recycling tag’allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for β‐lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily

N‐acetylglucosamine 1‐phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide‐activated UDP‐GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 Å resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long α‐helical arm: (i) an N‐terminal domain which resembles the dinucleotide‐binding Rossmann fold; and (ii) a C‐terminal domain which adopts a left‐handed parallel β‐helix structure (LβH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LβH domains, the long α‐helical linkers being seated on top of the arrangement and the N‐terminal domains projected away from the 3‐fold axis. In addition, the 2.3 Å resolution structure of the GlmU–UDP‐GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three‐dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP‐sugar bacterial pyrophosphorylases.

Synergistic enhancement of Toll-like receptor responses by NOD1 activation

NOD1 is an intracellular pattern‐recognition receptor specific for Gram‐negative peptidoglycan that is important in host response to infections (e.g. Helicobacter pylori and Shigella flexneri). Genetic variation in NOD1 predisposes to asthma and inflammatory bowel disease. Functional responses have not previously been studied in primary human cells. NOD1 activation by low nanomolar concentrations of the specific muropeptide ligand M‐TriDAP induced minimal human peripheral blood mononuclear cell TNF‐α, IL‐1β or IL‐10 secretion, but synergistically increased Toll‐like receptor (TLR)‐induced responses. Synergistic responses were seen across multiple ligands (to TLR1/2, 2/6, 4, 5, 7/8) and a broad range of cytokine secretion (TNF‐α, IL‐1α, IL‐1β, IL‐4, IL‐6, IL‐10, GM‐CSF). Synergy was also observed in the allogeneic mixed lymphocyte reaction. These responses were similar in cells homozygous for Crohns disease‐associated NOD2 mutations. In contrast to cell lines, primary human peripheral blood mononuclear cells respond to NOD1 muropeptides at ∼ 100‐fold lower concentrations. Cross‐talk between cytosolic NOD1 and membrane‐bound TLR enhances responses to the multiple antigens simultaneously presented by a microbe.

Peptidoglycan Molecular Requirements Allowing Detection by the Drosophila Immune Deficiency Pathway

Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor.

Murine Nod1 but not its human orthologue mediates innate immune detection of tracheal cytotoxin

Tracheal cytotoxin (TCT) was originally described as the minimal effector that was able to reproduce the cytotoxic response of Bordetella pertussis on ciliated epithelial cells. This molecule triggers pleiotropic effects such as immune stimulation or slow‐wave sleep modulation. Further characterization identified TCT as a specific diaminopimelic acid (DAP)‐containing muropeptide, GlcNAc‐(anhydro)MurNAc‐L‐Ala‐D‐Glu‐mesoDAP‐D‐Ala. Here, we show that the biological activity of TCT depends on Nod1, an intracellular sensor of bacterial peptidoglycan. However, Nod1‐dependent detection of TCT was found to be host specific, as human Nod1 (hNod1) poorly detected TCT, whereas mouse Nod1 (mNod1) did so efficiently. More generally, hNod1 required a tripeptide (L‐Ala‐D‐Glu‐mesoDAP) for efficient sensing of peptidoglycan, whereas mNod1 detected a tetrapeptide structure (L‐Ala‐D‐Glu‐mesoDAP‐D‐Ala). In murine macrophages, TCT stimulated cytokine secretion and NO production through Nod1. Finally, in vivo, injection of the tetrapeptide structure in mice triggered a transient yet strong release of cytokines into the bloodstream and the maturation of macrophages, in a Nod1‐dependent manner. This study thereby identifies Nod1 as the long sought after sensor of TCT in mammals.

Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis

The two‐dimensional membrane topology of the Escherichia coli and Staphylococcus aureus MraY transferases, which catalyse the formation of the first lipid intermediate of peptidoglycan synthesis, was established using the β‐lactamase fusion system. All 28 constructed mraY–blaM fusions produced hybrid proteins. Analysis of the ampicillin resistance of the strains with hybrids led to a common topological model possessing 10 transmembrane segments, five cytoplasmic domains and six periplasmic domains including the N‐ and C‐terminal ends. The agreement between the topologies of E. coli and S. aureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequences strongly suggested the validity of the model for all eubacterial MraYs. The primary structure of the 10 transmembrane segments diverged among orthologues, but they retained their hydrophobicity, number and size. The similarity of the sequences and distribution of the five cytoplasmic domains in both models, as well as their conservation among the MraY orthologues, clearly suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untruncated mraY restored growth. It was noteworthy that S. aureus MraY was functional in E. coli. An increased MraY transferase activity was observed only with the untruncated hybrids from both organisms.

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.

Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli.

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106–30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant ΔbacA,ΔybjG,ΔpgpB in which the expression of bacA is impaired at 42 °C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.