Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2

Nod1 and Nod2 are mammalian proteins implicated in the intracellular detection of pathogen-associated molecular patterns. Recently, naturally occurring peptidoglycan (PG) fragments were identified as the microbial motifs sensed by Nod1 and Nod2. Whereas Nod2 detects GlcNAc-MurNAc dipeptide (GM-Di), Nod1 senses a unique diaminopimelate-containing GlcNAc-MurNAc tripeptide muropeptide (GM-TriDAP) found mostly in Gram-negative bacterial PGs. Because Nod1 and Nod2 detect similar yet distinct muropeptides, we further analyzed the molecular sensing specificity of Nod1 and Nod2 toward PG fragments. Using a wide array of natural or modified muramyl peptides, we show here that Nod1 and Nod2 have evolved divergent strategies to achieve PG sensing. By defining the PG structural requirements for Nod1 and Nod2 sensing, this study reveals how PG processing and modifications, either by host or bacterial enzymes, may affect innate immune responses.     

Requirements of peptidoglycan structure that allow detection by the Drosophila Toll pathway

The Drosophila immune system is able to discriminate between classes of bacteria. Detection of Gram-positive bacteria involves a complex of two pattern recognition receptors: peptidoglycan recognition protein SA (PGRP-SA) and Gram-negative binding protein 1 (GNBP1). These activate the Toll signalling pathway. To define the cell wall components sensed by the host, we used highly purified peptidoglycan fragments of two principal Gram-positive bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae. We report that in both peptidoglycans, the minimal structure needed to activate the Toll pathway is a muropeptide dimer and that the free reducing end of the N-acetyl muramic acid residues of the muropeptides is essential for activity. Monomeric muropeptides were inactive and inhibitory in combination with dimers. Finally, peptidoglycan was degraded by the haemolymph of wild-type but not GNBP1 mutant flies. We suggest a model whereby GNBP1 is involved in the hydrolysis of Gram-positive peptidoglycan producing new glycan reducing ends, which are subsequently detected by PGRP-SA.     

Analysis of peptidoglycan structure from vegetative cells of Bacillus subtilis 168 and role of PBP 5 in peptidoglycan maturation.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilis vegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in the dacA mutant. This major low-molecular-weight PBP (DD-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.     

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation.     

All hands on deck—the role of chloroplasts, endoplasmic reticulum, and the nucleus in driving plant innate immunity

Plant innate immunity is mediated by cell membrane and intracellular immune receptors that function in distinct and overlapping cell-signaling pathways to activate defense responses. It is becoming increasingly evident that immune receptors rely on components from multiple organelles for the generation of appropriate defense responses. This review analyzes the defense-related functions of the chloroplast, nucleus, and endoplasmic reticulum (ER) during plant innate immunity. It details the role of the chloroplasts in synthesizing defense-specific second messengers and discusses the retrograde signal transduction pathways that exist between the chloroplast and nucleus. Because the activities of immune modulators are regulated, in part, by their subcellular localization, the review places special emphasis on the dynamics and nuclear–cytoplasmic transport of immune receptors and regulators and highlights the importance of this process in generating orderly events during an innate immune response. The review also covers the recently discovered contributions of the ER quality-control pathways in ensuring the signaling competency of cell surface immune receptors or immune regulators.     

Detailed structural analysis of the peptidoglycan of the human pathogen Neisseria meningitidis

We used reverse-phase high pressure liquid chromatography (HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and post source decay analysis (MALDI-PSD) to determine the muropeptide composition of the human pathogen Neisseria meningitidis. Structural assignment was determined for 28 muropeptide species isolated after HPLC separation and purification. Fourteen of these muropeptides were O-acetylated to different degrees. We identified the entire O-acetylation spectrum of dimers and trimers both in muropeptides and 1,6-anhydromuropeptides. On average, one of three disaccharides was O-acetylated. Furthermore, the degree of cross-linking of the N. meningitidis peptidoglycan was around 39% in all the strains analyzed. MALDI-PSD analysis of several muropeptide species indicated that meningococci only synthesize D-alanyl-meso-diaminopimelate cross-bridges. No muropeptides representative of covalent linkages of lipoproteins to the peptidoglycan could be identified, unlike in Escherichia coli. Finally, comparison of the muropeptide composition of penicillin-susceptible and penicillin-intermediate clinical strains of meningococci showed a positive correlation between the minimum inhibitory concentration (MIC) of penicillin G and the amount of muropeptides carrying an intact pentapeptide chain in the peptidoglycan. This suggests that reduced susceptibility to penicillin G in N. meningitidis is associated with a decrease in d,d-carboxypeptidase activity and/or D,D-transpeptidase activity.     

Mitochondria-derived reactive oxygen species negatively regulates immune innate signaling pathways triggered by a DNA virus, but not by an RNA virus

Reactive oxygen species (ROS) are crucial secondary messengers of signaling pathways. Redox-dependent signaling events have been previously described in the innate immune response. However, the mechanism by which ROS modulates anti-viral innate immune signaling is not fully clarified. Here, we report that mitochondria-derived ROS differentially regulate the innate response to DNA and RNA viruses (herpes simplex virus (HSV) and Sendai virus (SeV), respectively), with the cytokine response to HSV being negatively regulated by mitochondrial ROS. Importantly, specific activation of Toll-like receptors (TLRs) and DNA receptors (DNARs) but not retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), led to signaling cascades that were inhibited by mitochondrial ROS production. Thus, localized mitochondrial ROS exerts negative modulation of innate immune responses to the DNA virus HSV-2 but not the RNA virus SeV.     

Mammalian NLR proteins; discriminating foe from friend

Eukaryotic organisms of the plant and animal kingdoms have developed evolutionarily conserved systems of defence against microbial pathogens. These systems depend on the specific recognition of microbial products or structures by molecules of the host innate immune system. The first mammalian molecules shown to be involved in innate immune recognition of, and defence against, microbial pathogens were the Toll-like receptors (TLRs). These proteins are predominantly but not exclusively located in the transmembrane region of host cells. Interestingly, mammalian hosts were subsequently found to also harbour cytosolic proteins with analogous structures and functions to plant defence molecules. The members of this protein family exhibit a tripartite domain structure and are characterized by a central nucleotide-binding oligomerization domain (NOD). Moreover, in common with TLRs, most NOD proteins possess a C-terminal leucine-rich repeat (LRR) domain, which is required for the sensing of microbial products and structures. Recently, the name 'nucleotide-binding domain and LRR' (NLR) was coined to describe this family of proteins. It is now clear that NLR proteins play key roles in the cytoplasmic recognition of whole bacteria or their products. Moreover, it has been demonstrated in animal studies that NLRs are important for host defence against bacterial infection. This review will particularly focus on two subfamilies of NLR proteins, the NODs and 'NALPs', which specifically recognize bacterial products, including cell wall peptidoglycan and flagellin. We will discuss the downstream signalling events and host cell responses to NLR recognition of such products, as well as the strategies that bacterial pathogens employ to trigger NLR signalling in host cells. Cytosolic recognition of microbial factors by NLR proteins appears to be one mechanism whereby the innate immune system is able to discriminate between pathogenic bacteria ('foe') and commensal ('friendly') members of the host microflora.     

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli

Currently, reversed-phase high-performance liquid chromatography (HPLC) is the method of choice for determining the types and amounts of muropeptide subunits comprising bacterial peptidoglycan. Although effective and sensitive, the technique does not lend itself to high throughput screening, and its complexity and equipment requirements may dissuade some investigators from pursuing certain types of cell wall experiments. Previously, we showed that muropeptides can be labeled with a fluorescent dye and separated by fluorophore-assisted carbohydrate electrophoresis (FACE), a simple and rapid gel procedure that might serve as a prelude to more intense analysis by HPLC. To validate the utility of FACE, we used both techniques to perform a side-by-side analysis of the peptidoglycan of eight mutants and their Escherichia coli parent strain. FACE and HPLC both detected the seven major muropeptides, which represent more than 95% of the total muropeptides present in this organism. In addition, FACE returned the same relative and quantitative results in 92% of 72 measurements, indicating that the procedure gives an accurate overview of peptidoglycan composition. The results also suggest a possible biochemical activity for the AmpC and AmpH proteins of E. coli, and the use of FACE as an in vitro enzyme assay detected possible substrate preferences for the endopeptidase penicillin binding protein 4.     

Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca(2+), nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca(2+) into cells and provide a model linking this Ca(2+) current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca(2+) current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen-associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca(2+) current, which may be linked to NO generation due to buildup of cytosolic Ca(2+)/calmodulin.     

Nod1 participates in the innate immune response to Pseudomonas aeruginosa

The mammalian innate immune system recognizes pathogen-associated molecular patterns through pathogen recognition receptors. Nod1 has been described recently as a cytosolic receptor that detects specifically diaminopimelate-containing muropeptides from Gram-negative bacteria peptidoglycan. In the present study we investigated the potential role of Nod1 in the innate immune response against the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that Nod1 detects the P. aeruginosa peptidoglycan leading to NF-kappaB activation and that this activity is diminished in epithelial cells expressing a dominant-negative Nod1 construct or in mouse embryonic fibroblasts from Nod1 knock-out mice infected with P. aeruginosa. Finally, we demonstrate that the cytokine secretion kinetics and bacterial killing are altered in Nod1-deficient cells infected with P. aeruginosa in the early stages of infection.     

A novel role of the lumican core protein in bacterial lipopolysaccharide-induced innate immune response

Lumican is an extracellular matrix protein modified as a proteoglycan in some tissues. The core protein with leucine-rich repeats, characteristic of the leucine-rich-repeat superfamily, binds collagen fibrils and regulates its structure. In addition, we believe that lumican sequestered in the pericellular matrix interacts with cell surface proteins for specific cellular functions. Here we show that bacterial lipopolysaccharide sensing by the Toll-like receptor 4 signaling pathway and innate immune response is regulated by lumican. Primary cultures of lumican-deficient (Lum(-/-)) macrophages show impaired innate immune response to lipopolysaccharides with lower induction of tumor necrosis factor alpha (TNFalpha) and interleukin-6. Macrophage response to other pathogen-associated molecular patterns is not adversely affected by lumican deficiency, suggesting a specific role for the lumican core protein in the Toll-like receptor 4 pathway. An exogenous recombinant lumican core protein increases lipopolysaccharide-mediated TNFalpha induction and partially rescues innate immune response in Lum(-/-) macrophages. We further show that the core protein binds lipopolysaccharide. Immunoprecipitation of lumican from peritoneal lavage co-precipitates CD14, a cell surface lipopolysaccharide-binding protein that is involved in its presentation to Toll-like receptor 4. The Lum(-/-) mice are hypo-responsive to lipopolysaccharide-induced septic shock, with poor induction of pro-inflammatory cytokines, TNFalpha, and interleukins 1beta and 6 in the serum. Taken together, the data indicates a novel role for lumican in the presentation of bacterial lipopolysaccharide to CD14 and host response to this bacterial endotoxin.     

Primary structure of cyanelle peptidoglycan of Cyanophora paradoxa: a prokaryotic cell wall as part of an organelle envelope.

The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometry and amino acid analysis revealed N-acetylputrescine to be the substituent (E. Pittenauer, E. R. Schmid, G. Allmaier, B. Pfanzagl, W. Löffelhardt, C. Quintela, M. A. de Pedro, and W. Stanek, Biol. Mass Spectrom. 22:524-536, 1993). In addition to the 4 monomers already known, 8 dimers, 11 trimers, and 6 tetramers were characterized. An average glycan chain length of 51 disaccharide units was determined by the transfer of [U-14C]galactose to the terminal N-acetylglucosamine residues of cyanelle peptidoglycan. The muropeptide pattern is discussed with respect to peptidoglycan biosynthesis and processing.     

Functional analysis of Streptococcus pneumoniae MurM reveals the region responsible for its specificity in the synthesis of branched cell wall peptides

The recently identified murMN operon of Streptococcus pneumoniae encodes enzymes involved in the synthesis of branched structured muropeptides of the pneumococcal cell wall peptidoglycan. Its inactivation was shown to cause production of a peptidoglycan composed exclusively of linear muropeptides and a virtually complete loss of resistance in penicillin-resistant strains. The studies described in this communication follow up these observations in several directions. The substrate of the MurM-catalyzed reaction (addition of alanine or serine) was identified as the lipid-linked N-acetylglucosamine-muramyl pentapeptide. Different murM alleles from several penicillin-resistant S. pneumoniae strains, each with a characteristic branched peptide pattern, were cloned into pLS578, a pneumococcal plasmid capable of replicating in S. pneumoniae, and transformed into the penicillin-susceptible laboratory strain R36A. All transformants remained penicillin-susceptible; however, their cell wall composition changed in directions corresponding to the muropeptide pattern of the strain from which the murM allele was derived. This suggests that the muropeptide composition of the pneumococcal cell walls is determined by the particular murM allele carried by the cells. A 30-amino acid long sequence within the MurM protein was shown to be the main determinant of the specificity of the reaction (addition of alanine versus serine).     

Self-consuming innate immunity in Arabidopsis

Programmed cell death (PCD) associated with the pathogen-induced hypersensitive response (HR) is a hallmark of plant innate immunity. HR PCD is triggered upon recognition of pathogen effector molecules by host immune receptors either directly or indirectly via effector modulation of host targets. However, it has been unclear by which molecular mechanisms plants execute PCD during innate immune responses. We recently examined HR PCD in autophagy-deficient Arabidopsis knockout mutants (atg) and find that PCD conditioned by one class of plant innate immune receptors is suppressed in atg mutants. Intriguingly, HR triggered by another class of immune receptors with different genetic requirements is not compromised, indicating that only a specific subset of immune receptors engage the autophagy pathway for HR execution. Thus, our work provides a primary example of autophagic cell death associated with innate immune responses in eukaryotes as well as of pro-death functions for the autophagy pathway in plants.     

Role of Nods in bacterial infection

Research into intracellular sensing of microbial products is an up and coming field in innate immunity. Nod1 and Nod2 are members of the rapidly expanding family of NACHT domain-containing proteins involved in intracellular recognition of bacterial products. Nods proteins are involved in the cytosolic detection of peptidoglycan motifs of bacteria, recognized through the LRR domain. The role of the NACHT-LRR system of detection in innate immune responses is highlighted at the mucosal barrier, where most of the membranous Toll like receptors (TLRs) are not expressed, or with pathogens that have devised ways to escape TLR sensing. For a given pathogen, the sum of the pathways induced by the recognition of the different "pathogen associated molecular patterns" (PAMPs) by the different pattern recognition receptors (PRRs) trigger and shape the subsequent innate and adaptive immune responses. Knowledge gathered during the last decade on PRR and their agonists, and recent studies on bacterial infections provide new insights into the immune response and the pathogenesis of human infectious diseases.     

The role of innate immune receptors in the control of Brucella abortus infection: toll-like receptors and beyond

Research into intracellular sensing of microbial products is an up and coming field in innate immunity. Toll-like receptors (TLRs) recognize Brucella spp. and bacterial components and initiate mononuclear phagocyte responses that influence both innate and adaptive immunity. Recent studies have revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella infection. TLR2, TLR4 and TLR9 have been implicated in host interactions with Brucella; however, TLR9 has the most prominent role. Further, the relationship between specific Brucella molecules and various signal transduction pathways needs to be better understood. MyD88-dependent and TRIF-independent signaling pathways are involved in Brucella activation of innate immune cells through TLRs. We have recently reported the critical role of MyD88 molecule in dendritic cell maturation and interleukin-12 production during B. abortus infection. This article discusses recent studies on TLR signaling and also highlights the contribution of NOD and type I IFN receptors during Brucella infection. The better understanding of the role by such innate immune receptors in bacterial infection is critical in host-pathogen interactions.