Structural and immunochemical relatedness suggests a conserved pathogenicity motif for secondary cell wall polysaccharides in Bacillus anthracis and infection-associated Bacillus cereus.

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-ß-d-ManpNAc-(1→4)-ß-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and ß-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying "anthrax-like" symptoms in Cameroon (Bc CA) and Côte d'Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10-20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.

Structural characterization of the immunostimulatory exopolysaccharide produced by Leuconostoc mesenteroides strain NTM048.

The exopolysaccharide (EPS) produced by probiotic Leuconostoc mesenteroides subsp. mesenteroides strain NTM048 has been reported to be an immunostimulant that enhances mucosal IgA production. In this study, we found that intranasal administration of mice with the EPS and an antigen (ovalbumin) resulted in secretion of antigen-specific IgA and IgG in the airway mucosa and the serum, suggesting that the EPS has the adjuvant activity for use with mucosal vaccination. Methylation analysis coupled to GC-MS, and 1D and 2D NMR spectroscopy revealed that 94% of the EPS consists of an α-(1 â†’ 6) glucan containing 4% of 1→3-linked α-glucose branches. To determine structures of minor components, we enzymatically digested the glucan with dextranase and used 2D NMR spectroscopy to identify the remaining polymer as a fructan (or fructans), containing both ß-(2 â†’ 6)- and ß-(2 â†’ 1)-linked fructofuranose residues. These residues may either enter into separate polymers of each linkage type or form a mixed fructan containing both linkage types.

SpyB, a Small Heme-Binding Protein, Affects the Composition of the Cell Wall in Streptococcus pyogenes.

Streptococcus pyogenes (Group A Streptococcus or GAS) is a hemolytic human pathogen associated with a wide variety of infections ranging from minor skin and throat infections to life-threatening invasive diseases. The cell wall of GAS consists of peptidoglycan sacculus decorated with a carbohydrate comprising a polyrhamnose backbone with immunodominant N-acetylglucosamine side-chains. All GAS genomes contain the spyBA operon, which encodes a 35-amino-acid membrane protein SpyB, and a membrane-bound C3-like ADP-ribosyltransferase SpyA. In this study, we addressed the function of SpyB in GAS. Phenotypic analysis of a spyB deletion mutant revealed increased bacterial aggregation, and reduced sensitivity to ß-lactams of the cephalosporin class and peptidoglycan hydrolase PlyC. Glycosyl composition analysis of cell wall isolated from the spyB mutant suggested an altered carbohydrate structure compared with the wild-type strain. Furthermore, we found that SpyB associates with heme and protoporphyrin IX. Heme binding induces SpyB dimerization, which involves disulfide bond formation between the subunits. Thus, our data suggest the possibility that SpyB activity is regulated by heme.

Modification of Helicobacter pylori Peptidoglycan Enhances NOD1 Activation and Promotes Cancer of the Stomach.

Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan into host cells. NOD1 is an intracellular immune receptor that senses peptidoglycan 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 peptidoglycan 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. Coculture 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, preactivation of NOD1 before bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared with noncancer samples harvested from the same patients. These results indicate that peptidoglycan 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.

Noncanonical inflammasome activation by intracellular LPS independent of TLR4.

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.

Helicobacter pylori peptidoglycan modifications confer lysozyme resistance and contribute to survival in the host.

UNLABELLED: 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 rkp-1 cluster is required for secretion of Kdo homopolymeric capsular polysaccharide in Sinorhizobium meliloti strain Rm1021.

Under conditions of nitrogen stress, leguminous plants form symbioses with soil bacteria called rhizobia. This partnership results in the development of structures called root nodules, in which differentiated endosymbiotic bacteria reduce molecular dinitrogen for the host. The establishment of rhizobium-legume symbioses requires the bacterial synthesis of oligosaccharides, exopolysaccharides, and capsular polysaccharides. Previous studies suggested that the 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) homopolymeric capsular polysaccharide produced by strain Sinorhizobium meliloti Rm1021 contributes to symbiosis with Medicago sativa under some conditions. However, a conclusive symbiotic role for this polysaccharide could not be determined due to a lack of mutants affecting its synthesis. In this study, we have further characterized the synthesis, secretion, and symbiotic function of the Kdo homopolymeric capsule. We showed that mutants lacking the enigmatic rkp-1 gene cluster fail to display the Kdo capsule on the cell surface but accumulate an intracellular polysaccharide of unusually high M(r). In addition, we have demonstrated that mutations in kdsB2, smb20804, and smb20805 affect the polymerization of the Kdo homopolymeric capsule. Our studies also suggest a role for the capsular polysaccharide in symbiosis. Previous reports have shown that the overexpression of rkpZ from strain Rm41 allows for the symbiosis of exoY mutants of Rm1021 that are unable to produce the exopolysaccharide succinoglycan. Our results demonstrate that mutations in the rkp-1 cluster prevent this phenotypic suppression of exoY mutants, although mutations in kdsB2, smb20804, and smb20805 have no effect.

Oxidative stress-induced peptidoglycan deacetylase in Helicobacter pylori.

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 PG-deacetylated 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.

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides.

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp.

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.