Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4(-/-) mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

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Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH(2)-(1→.

Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1 → 4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and β-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000 Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.

Structural modification of peptidoglycan (PG) is one of the mechanisms that pathogenic bacteria use to evade the host innate immune system. For the noninvasive human gastric pathogen Helicobacter pylori, PG delivery to the host cells is one trigger of the immune response. H. pylori HP310 was markedly up-expressed upon cell exposure to oxidative stress. However, disruption of HP310 did not produce a phenotype distinguishable from the parent, including oxidative stress resistance characteristics. HP310 shows very weak homology to a known gene pgdA encoding PG deacetylase in Streptococcous pneumoniae. PGs from wild type H. pylori and the HP310 mutant were purified and analyzed by matrix-assisted laser desorption ionization time-of-flight and high pressure liquid chromatography. The parent strain PG is partially deacetylated, whereas several major PGdeacetylated muropeptides are absent or significantly reduced in the HP310 mutant. PG deacetylase activity was directly demonstrated by use of pure PG and HP310 protein by measuring the release of acetic acid. The Gram-negative bacterium H. pylori is highly resistant to lysozyme (up to 50 mg/ml), but the HP310 mutant is less resistant to lysozyme compared with the parent strain. Complementation of an hp310 strain with the wild type gene restored lysozyme resistance. The purified PG from the mutant is more susceptible to lysozyme (0.3 mg/ml) digestion than the wild type PG. The PG deacetylation appears to confer lysozyme resistance to escape immune detection. HP310 is representative of a new subfamily of bacterial PG deacetylases.Helicobacter pylori, a pathogenic bacterium infecting over 50% of humans, is the etiological agent for gastritis, peptic ulcer, and gastric cancer (1). H. pylori is highly adapted to its ecologic niche, the human gastric mucosa. The pathogenesis of H. pylori relies on its persistence in surviving a harsh environment, including acidity, peristalsis, and attack by phagocyte cells and their released reactive oxygen species (ROS) 2 (2). H. pylori has a unique array of features that permit entry into the mucus, attachment to epithelial cells, evasion of the immune response, and as a result, persistent colonization and transmission. Numerous virulence factors in H. pylori have been extensively studied, including urease, flagella, BabA adhesin, the vacuolating cytotoxin (VacA), and the cag pathogenicity island (cag-PAI). Peptidoglycan (PG) was found in recent years to be an important factor involved in virulence by pathogenic bacteria (3). PG is one of the main protective barriers in the bacterial cell wall. PG consists of glycan strands made of alternating ␤-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptide chains. In mammalian cells, a group of pattern recognition molecules (Nod1 and Nod2) can sense bacterial PG degradation products (muropeptides), initiating innate immune responses (4, 5). For example, human Nod1 (Toll-like receptor) specifically detects a unique (GlcNAc-MurNAc) tripept...

Helicobacter pylori is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan (PGN) into host cells. NOD1 is an intracellular immune receptor that senses PGN from Gram-negative bacteria and responds by inducing autophagy and activating NF-κB, leading to inflammation-mediated bacterial clearance; however chronic pathogens can evade NOD1-mediated clearance by altering PGN structure. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA; therefore, we sought to define the role of H. pylori PgdA in NOD1-dependent activation of NF-κB, inflammation, and cancer. Co-culture of H. pylori strain 7.13 or its pgdA− isogenic mutant with AGS gastric epithelial cells or HEK293 epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection of Mongolian gerbils with an H. pylori pgdA− mutant strain led to significantly decreased levels of inflammation and malignant lesions in the stomach; however, pre-activation of NOD1 prior to bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared to non-cancer samples harvested from the same patients. These results indicate that PGN deacetylation plays an important role in modulating host inflammatory responses to H. pylori, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche.

The prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized a Helicobacter pylori PG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that a patA strain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA in H. pylori PG O-acetylation. The PG modification mutant strains (pgdA, patA, or pgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity to H. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). The pgdA patA double mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from a pgdA patA mutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.Importance Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japon&um, whose product is required for the addition of a 2-0-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.Leguminous plants are infected (nodulated) by the gramnegative, soil bacteria Rhizobium, Azorhizobium, and Bradyrhizobium species, which establish a nitrogen-fixing symbiosis within the roots. There is now considerable information available concerning the genetic traits of these bacteria that are required for the establishment of nitrogen-fixing symbiosis. A number of nodulation (nod, nol) genes have been identified (48). These genes fall into two general groups: the common nod genes (e.g., nodABC) that are essential for nodulation of any host and the host specificity genes that appear to determine specific nodulation of a narrow range of hosts (reviewed in references 13, 14, and 21). Recent evidence indicates that the function of many, if not all, of the nod genes is to synthesize or modify lipooligosaccharide signal molecules that initiate many of the initial nodulation responses in the plant (13, 14, 55). For example, Rhizobium meliloti has been shown to synthesize a sulfated lipooligosaccharide that induces many of the early events of nodulation when applied to alfalfa roots (13,14). Recent results indicate that the protein products of the host specificity genes of R. meliloti, nodHPQ, are involved in the specific sulfation reaction leading to the synthesis of NodRm-1 (e.g., see references 4, 41, and 50). We recently identified the lipooligosaccharide nodulation signals produced by Bradyrhizobium japonicum USDA110 and USDA135 and Bradyrhizobium elkanii USDA61 (9,47 NodD (6,25,33