Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0348321 (Haemophilus)
 15,372 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The resistance of gonococci in most patients to complement mediated killing by human serum is due to sialylation of their lipopolysaccharide (LPS) which prevents bactericidal antibody from reacting with target sites. Two of the host factors responsible are: cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA), a well-known sialylating agent, and another factor which enhances the transfer of sialyl groups from CMP-NANA to LPS catalysed by a gonococcal sialyltransferase. The bacterial determinant of resistance is a conserved LPS component of about 4.5 kDa which is sialylated at a terminal Gal beta 1-4GlcNAc site on its side chain. The sialylated LPS forms a surface coat which is stainable by ruthenium red and connected with previously described 'capsules'. These observations sparked off an explosion of research. Recent publications show that sialylation of LPS by CMP-NANA affects additional important aspects of gonococcal pathogenicity, notably interactions with antibodies and phagocytes, and rendering the gonococcal surface more 'host-like'. Also, the observations have prompted an examination of LPS from some other pathogens for the presence of sialyl groups with positive results for Neisseria meningitidis and Haemophilus influenzae.
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PMID:The sialylation of gonococcal lipopolysaccharide by host factors: a major impact on pathogenicity. 147 64

Immunochemical studies of the lipo-oligosaccharides (LOS) of the Gram-negative bacteria Neisseria gonorrhoeae and Neisseria meningitidis have revealed some interesting structural characteristics of these LOS that might relate to their roles during pathogenesis. The carbohydrate moieties of the LOS of pathogenic Neisseria mimic carbohydrates present in glycosphingolipids of human cells. Firstly, an LOS component present among a number of Neisseria species is antigenically and/or chemically identical to lactoneoseries glycosphingolipids present in human cells. The lactoneoseries LOS becomes sialylated on Neisseria gonorrhoeae when they are grown in the presence of cytidine 5'-monophospho-N-acetyl-neuraminic acid (CMP-NANA), the nucleotide sugar for sialic acid. Examination of gonococci present in exudates from males with natural infection indicates that sialylation also occurs in vivo. The mechanism for this process apparently involves a bacterial sialyltransferase scavenging available host CMP-NANA ("host-modification" of LOS) and transferring the sialic acid to the lactoneoserieslike LOS. Strains of N. meningitidis and Haemophilus influenzae also express similarly sialylated LOS suggesting that this is a common mechanism of pathogenesis among these bacteria. Additional examples of LOS that mimic other glycosphingolipid series have been identified also and the fact that multiple series can be expressed in a single population of gonococci suggests that a diverse set of LOS can be presented to the host during infection. It is possible that this diverse set of LOS serve different functions for the bacteria in various hosts and/or environments during infection.
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PMID:Lipo-oligosaccharides (LOS) of mucosal pathogens: molecular mimicry and host-modification of LOS. 833 Sep 4

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.
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PMID:Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide. 839 87

Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)- at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)- at m/z 1703.9. The identification of the mono-functional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis.
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PMID:A mono-functional 3-deoxy-D-manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influenzae. 919 66

The presence of sialic acid as a component of cell surface lipooligosaccharides or capsular polysaccharides has been shown to be correlated with the virulence of a number of Gram-negative mucosal pathogens, including several Haemophilus and Neisseria spp. As part of our efforts to evaluate the role of sialic acid in the pathobiology of these organisms, we have initiated studies of the enzymes from Haemophilus ducreyi (the infectious agent of chancroid) responsible for the activation and attachment of sialic acid to the lipooligosaccharide. In this report, we describe results of an investigation of the steady-state kinetic mechanism of the activating enzyme, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. Using a combination of initial velocity, product inhibition, and dead-end inhibition studies, the reaction is shown to be freely reversible and to proceed through an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuAc dissociates last. In addition, a detailed analysis of the kinetic expressions for the observable constants is presented showing how the variation in apparent product inhibition constants (Kii) can be used to predict the rate-limiting step in kcat, which appears to be dissociation of CMP-NeuAc in this enzyme. To our knowledge, this relationship has not been previously recognized.
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PMID:Investigation of the kinetic mechanism of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate-limiting steps from product inhibition analysis. 1032 Mar 48

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.
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PMID:Novel method for enzymatic synthesis of CMP-NeuAc. 1157 11

Haemophilus somnus isolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase of H. somnus strain 738 was confirmed to preferentially sialylate the Gal(beta)-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by enzyme immunoassay and Western blotting. Furthermore, sialylation of the LOS enhanced the resistance of H. somnus to the bactericidal action of antiserum to LOS. Sialylation and increased resistance to killing by normal serum also occurred in a deletion mutant that was deficient in the terminal Gal-GlcNAc disaccharide. LOS sialylation may therefore be an important virulence mechanism to protect H. somnus against the host immune system.
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PMID:Incorporation of N-acetylneuraminic acid into Haemophilus somnus lipooligosaccharide (LOS): enhancement of resistance to serum and reduction of LOS antibody binding. 1218 31

The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-manno-octulosonate (KDO) by forming CMP-KDO. It is essential for the biosynthesis of lipopolysaccharides in Gram-negative bacteria and is a potential target for the discovery of antibacterial agents. L-CKS from Haemophilus influenzae was overexpressed with a C-terminal hexahistidine tag in Escherichia coli and crystallized in the presence of the substrate KDO at 297 K using PEG 4000 as a precipitant and ethylene glycol as an additive. The diffraction limit and spot shape of the native crystal could be improved significantly by dehydration/annealing. X-ray diffraction data were collected to 2.5 A resolution from a native crystal. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2(1), with unit-cell parameters a = 48.6, b = 83.1, c = 117.3 A. The presence of two monomers of recombinant L-CKS in the crystallographic asymmetric unit gives a reasonable V(M) of 2.05 A(3) Da(-1), with a solvent content of 40.0%.
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PMID:Crystallization and preliminary X-ray crystallographic studies of 3-deoxy-manno-octulosonate cytidylyltransferase from Haemophilus influenzae. 1249 64

Otitis media, a common and often recurrent bacterial infection of childhood, is a major reason for physician visits and the prescription of antimicrobials. Haemophilus influenzae is the cause of approximately 20% of episodes of bacterial otitis media, but most strains lack the capsule, a factor known to play a critical role in the virulence of strains causing invasive H. influenzae disease. Here we show that in capsule-deficient (nontypeable) strains, sialic acid, a terminal residue of the core sugars of H. influenzae lipopolysaccharide (LPS), is a critical virulence factor in the pathogenesis of experimental otitis media in chinchillas. We used five epidemiologically distinct H. influenzae isolates, representative of the genetic diversity of strains causing otitis media, to inoculate the middle ear of chinchillas. All animals developed acute bacterial otitis media that persisted for up to 3 wk, whereas isogenic sialic acid-deficient mutants (disrupted sialyltransferase or CMP-acetylneuraminic acid synthetase genes) were profoundly attenuated. MS analysis indicated that WT bacteria used to inoculate animals lacked any sialylated LPS glycoforms. In contrast, LPS of ex vivo organisms recovered from chinchilla middle ear exudates was sialylated. We conclude that sialylated LPS glycoforms play a key role in pathogenicity of nontypeable H. influenzae and depend on scavenging the essential precursors from the host during the infection.
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PMID:Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media. 1285 65

The enzyme 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase; CKS) catalyzes the activation of 3-deoxy-D-manno-octulosonate (or 2-keto-3-deoxy-manno-octonic acid; KDO) by forming CMP-KDO. CKS is unique to Gram-negative bacteria and is an attractive target for the development of antibacterial agents. The crystal structure of CKS from Haemophilus influenzae in complex with the substrate KDO has been determined at 2.30 A resolution by combining single-wavelength anomalous diffraction and molecular-replacement methods. The two monomers in the asymmetric unit differ in the conformation of their C-terminal alpha-helix (Ala230-Asn254). The KDO bound to the active site exists as the beta-pyranose form in the (5)C(2) chair conformation. The structure of CKS from H. influenzae in complex with KDO will be useful in structure-based inhibitor design.
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PMID:Structure of 3-deoxy-manno-octulosonate cytidylyltransferase from Haemophilus influenzae complexed with the substrate 3-deoxy-manno-octulosonate in the beta-configuration. 1901 7


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