The biological response to endotoxin mediated through the Toll-like receptor 4 (TLR4)-MD-2 receptor complex is directly related to lipid A structure or configuration. Endotoxin structure may also influence activation of the MyD88-dependent and -independent signaling pathways of TLR4. To address this possibility, human macrophage-like cell lines (THP-1, U937, and MM6) or murine macrophage RAW 264.7 cells were stimulated with picomolar concentrations of highly purified endotoxins. Harvested supernatants from previously stimulated cells were also used to stimulate RAW 264.7 or 23ScCr (TLR4-deficient) macrophages (i.e., indirect induction). Neisseria meningitidis lipooligosaccharide (LOS) was a potent direct inducer of the MyD88-dependent pathway molecules tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3α (MIP-3α), and the MyD88-independent molecules beta interferon (IFN-β), nitric oxide, and IFN-γ-inducible protein 10 (IP-10). Escherichia coli 55:B5 and Vibrio cholerae lipopolysaccharides (LPSs) at the same pmole/ml lipid A concentrations induced comparable levels of TNF-α, IL-1β, and MIP-3α, but significantly less IFN-β, nitric oxide, and IP-10. In contrast, LPS from Salmonella enterica serovars Minnesota and Typhimurium induced amounts of IFN-β, nitric oxide, and IP-10 similar to meningococcal LOS but much less TNF-α and MIP-3α in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF-α release but did not influence nitric oxide release. IFN-β polyclonal antibody and IFN-α/β receptor 1 antibody significantly reduced nitric oxide release. N. meningitidis endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. E. coli 55:B5 and Vibrio cholerae LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while Salmonella LPS activated the MyD88-independent pathway.

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants ofNeisseria meningitidisthat contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS α or β chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-d-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an ∼10-fold (P< 0.0001) reduction in biologic activity compared to KDO₂-containing meningococcal LOS. Removal of KDO₂from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO₂linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae , though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae . To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient Δ cpsD strains. Compared to wild-type S. iniae , the Δ cpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l -fucose, d -mannose, d -galactose, d -glucose, d -glucuronic acid, N -acetyl- d -galactosamine, and N -acetyl- d -glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The Δ cpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae . Increased susceptibility of the S. iniae Δ cpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.

Lipopolysaccharide, lipooligosaccharide (LOS), or endotoxin is important in bacterial survival and the pathogenesis of gram-negative bacteria. A necessary step in endotoxin biosynthesis is 3-deoxy- d -manno-octulosonic acid (Kdo) glycosylation of lipid A, catalyzed by the Kdo transferase KdtA (WaaA). In enteric gram-negative bacteria, this step is essential for survival. A nonpolar kdtA :: aphA-3 mutation was created in Neisseria meningitidis via allelic exchange, and the mutant was viable. Detailed structural analysis demonstrated that the endotoxin of the kdtA :: aphA-3 mutant was composed of fully acylated lipid A with variable phosphorylation but without Kdo glycosylation. In contrast to what happens in other gram-negative bacteria, tetra-acylated lipid IV _A did not accumulate. The LOS structure of the kdtA :: aphA-3 mutant was restored to the wild-type structure by complementation with kdtA from N. meningitidis or Escherichia coli . The expression of a fully acylated, unglycosylated lipid A indicates that lipid A biosynthesis in N. meningitidis can proceed without the addition of Kdo and that KdtA is not essential for survival of the meningococcus.

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS 402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N -acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.