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)

Heme can serve Haemophilus influenzae as a source of both essential porphyrin and iron. In extracellular mammalian body fluids neither free heme nor free iron is available, since they are tightly bound to hemopexin and transferrin, respectively. Since H. influenzae grows in the presence of iron-transferrin and heme-hemopexin and is known to express a saturable receptor for transferrin, we investigated the process by which this pathogen acquired heme from hemopexin for use as an iron source. The ability of human and rabbit hemopexin to donate heme as a source of iron to H. influenzae type b strains was demonstrated by plate bioassays. With a dot enzyme assay with biotinylated hemopexin as ligand, H. influenzae bound heme-hemopexin and apo-hemopexin following growth in iron-restricted, but not in iron-sufficient, medium. Competitive binding studies with heme-hemopexin and apo-hemopexin demonstrated saturability of binding. Neither heme, protoporphyrin IX, hemoglobin, nor transferrin blocked the binding of hemopexin to whole cells, demonstrating the specificity of binding. Treatment of whole H. influenzae cells with trypsin abolished binding. Taken together, these observations suggest that H. influenzae type b expresses an outer membrane protein(s) which acts as a receptor for hemopexin and which is regulated by the availability of iron in the growth medium. In iron-restricted media, H. influenzae 706705 and DL42 did not express the 100-kDa hemopexin-binding protein previously reported (M.S. Hanson, S.E. Pelzel, J. Latimer, U. Muller-Eberhard, and E.J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). The putative iron-regulated hemopexin receptor was solubilized from cell envelopes of H. influenzae 706705, DL42, and Eagan with the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and isolated by affinity chromatography on heme-hemopexin-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins bound to the affinity resin revealed three proteins of 29, 38, and 57 kDa, of which the 57- and 29-kDa proteins bound hemopexin after Western blotting (immunoblotting). A monoclonal antibody to the 57-kDa hemopexin-binding protein of 706705 recognized a 57-kDa protein on Western blots of the cell envelope proteins of 706705, DL42, and Eagan; no reaction was observed with the 100-kDa hemopexin-binding protein of DL42. These data suggest that some H. influenzae strains possess at least two hemopexin receptors, the expression of which is determined by the prevailing growth environment.
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PMID:Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b. 826 49

Brazilian purpuric fever (BPF) is a newly described pediatric syndrome that results in significant morbidity and mortality. BPF is caused by specific phenotypic strains of Haemophilus influenzae biogroup aegyptius that are capable of intravascular survival. Immunoblotting of outer membrane proteins of H. influenzae biogroup aegyptius with normal human serum showed that most virulent strains of H. influenzae biogroup aegyptius associated with BPF expressed an immunologically prominent protein at 72 kDa. A corresponding protein in avirulent isolates migrated at 79 kDa. Although a minor component on SDS-PAGE analysis of the outer membrane, specific antibody against this protein is present in high concentrations in normal human serum.
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PMID:Expression of an immunoreactive 72 kDa protein in strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever. 830 58

Lipopolysaccharide (LPS) is a major determinant of Neisseria, meningitidis virulence. A key feature of meningococcal LPS is the phase-variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase-variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae, was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic-resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine-SDS-PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto-N-neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5'-end of the coding sequence. As the lacto-N-neotetraose structure in meningococcal LPS is subject to phase-variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an 'off' phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped-strand mispairing mechanism, operating in the first ORF, controls the phase-variable expression of lacto-N-neotetraose.
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PMID:Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis. 881 94

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemophilus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neisseria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)(n) encoded by multiple tandem repeats of 5'-CAAT-3'. This observation suggested that (SINQ)(n) might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5'-CAAT-3' repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)(n) is not a prerequisite for the enzymatic function of Lic2A and that the 5'-CAAT-3' repeats in lic2A function solely as a mechanism for generating phase variation. This observation suggested that wide variation in the number of 5'-CAAT-3' repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5'-CAAT-3' repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)(n) indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.
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PMID:Tandem repeats of the tetramer 5'-CAAT-3' present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae. 886 Dec 14

By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the IgtA-E genes.
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PMID:Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide. 901 Oct 46

The present study includes 178 Haemophilus influenzae strains isolated in different pediatric hospitals from Havana, Cuba, during 1991-1994, associated to divers infections (meningitis, respiratory sepsis, primary bacteremia). A combination of various typing and subtyping methods was used as epidemiological markers: serotyping (slide agglutination with diagnostical serum a-f and latex agglutination), biotyping according to Killian's procedures (by determination of indole production, urease and ornithine decarboxylase activity), subtyping by fermentative profiles according to Roberts' methods (glucose, maltose, xylose and fructose) and outer membrane protein profile subtyping (vesicles extraction by a modified Barenkamp's method, analysis by lineal and gradient SDS-PAGE and assessment according to our own classification system). Serotype b was identified in 89.3%, biotype I was the most frequent (79.1%), other biotypes (II, III, IV and V) were also identified. Fermentative profile D (glucose, maltose, xylose and fructose positive) was the most frequent (52.8%) while profile G (glucose, maltose, xylose positive and fructose negative) represented 20.2%. Other known profiles were present. PA2 (33.7%) was the most frequent OMP subtype. Even though 11 different protein subtypes were found, the 77.5% of the strains were located in only three OMP electrophoretic subtypes (PA2, PC1, LA2).
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PMID:[Utilization of different microbiological markers in the study of Haemophilus influenzae]. 902 20

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de-O-acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de-O-acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy-D-manno-octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS-PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.
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PMID:Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae. 904 90

Plasminogen binding proteins have been described both for Gram positive and Gram negative bacteria. In the present work we describe the purification and characterization of a plasminogen binding protein from Haemophilus influenzae (strain HI-23459). Bacteria were sonicated in order to solubilize plasminogen-binding proteins. The supernatant was subjected to affinity chromatography on plasminogen kringle-4 fragment bound to Sepharose 4B and subsequently processed by ion-exchange chromatography on DEAE-Sepharose CL-6B. Characterization of the protein by SDS-PAGE displayed a single band with a molecular mass of about 55,000, both prior to and after reduction. The purified protein stimulates tPA (tissue plasminogen activator) catalysed plasminogen activation by a factor of approximately 300, mainly due to a decrease in K(m). Antibodies were raised in rabbits and used in quantitative and qualitative analysis. However, using a FITC-conjugate we failed to demonstrate the presence of the purified protein on the surface of intact bacteria. The corresponding gene was isolated from a lambda EMBL3 phage library prepared from chromosomal DNA from the same H. influenzae strain, using an oligonucleotide probe based on the NH2-terminal amino acid sequence. An open reading frame corresponding to 472 amino acid was found. The amino acid sequence of the translated gene demonstrates 97% identity with the recently published sequence from aspartate ammonia lyase (aspartase) from H. influenzae. Enzymatic analysis of the purified protein revealed a high aspartase activity.
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PMID:Purification and characterisation of a plasminogen-binding protein from Haemophilus influenzae. Sequence determination reveals identity with aspartase. 909 5

Several flavoproteins and cytochromes that occur as major components in extracts of the yellow bioluminescence Y1 strain of the marine bacterium Vibrio fischeri have been purified and characterized with respect to their mass (SDS/PAGE and matrix-assisted laser-desorption/ionization MS), chromatographic properties, N-terminal sequence, and spectroscopy (absorption, fluorescence emission and anisotropy decay). The investigated proteins were as follows: yellow fluorescence protein (YFP) with bound riboflavin, FMN or 6,7-dimethyl-8-ribityllumazine; a blue fluorescence protein (BFP) with bound 6,7-dimethyl-8-ribityllumazine, riboflavin, or 6-methyl-7-oxo-8-ribityllumazine; thioredoxin reductase with FAD as ligand; and two c-type diheme cytochromes, c551 and c554. We present evidence that the riboflavin-bound YFP has an N-terminal sequence corresponding to that published for the dimeric YFP. We show that an equilibrium replacement of the riboflavin can be made with excess lumazine derivative and that lumazine-bound YFP has different bioluminescence properties to those of the lumazine protein from Photobacterium leiognathi. BFP is a different protein again, and in the bacterial lysate it occurs in multiple forms, ligated to either riboflavin, lumazine, or the 7-oxolumazine derivative. The N-terminal sequence for BFP shows similarities to those of the YFP proteins and to lumazine protein and riboflavin synthase from Photobacterium. BFP in any form has no bioluminescence or riboflavin-synthase activity. A 70-kDa fluorescent flavoprotein with FAD as ligand has an N-terminal sequence highly similar to those of thioredoxin reductases from Haemophilus influenzae and Escherichia coli. Cytochrome contaminations in previous preparations of YFP have been removed and are identified as the two c-type cytochromes c551 and c554. Both inhibit the NADH-induced bioluminescence in the reductase/luciferase system with the luciferases from P. leiognathi and V. fischeri. The N-terminal amino acid sequence of the cytochrome (c551) corresponds to a diheme cytochrome c4. The spectral properties of c554 are similar to those of other c5 cytochromes, and both c554 and c551 have absorption spectra similar to those of the respective cytochromes from the gram-negative bacteria Pseudomonas and Azotobacter.
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PMID:Purification and characterization of flavoproteins and cytochromes from the yellow bioluminescence marine bacterium Vibrio fischeri strain Y1. 918 20

Haemophilus parasuis malate dehydrogenase ((S)-malate:NAD+ oxidoreductase; EC 1.1.1.37) isolated from cell sonicates was purified 584-fold to electrophoretic homogeneity with a 19% recovery and a specific activity of 222 units/mg protein. SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the purified enzyme to be a dimer composed of 34,600 molecular weight subunits. Kinetic parameters for all four substrates in the forward and reverse reactions indicated a sequential mechanism for this enzymic process. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme and NADH the second product released. Protection against thermodenaturation of the enzyme by NAD and not by malate was supportive of this mechanism. A pronounced product inhibition by NADH (K(i) = 9.0 microM) was observed. Although NADP did not serve as a coenzyme, a number of analogs of NAD structurally altered in the nitrogen base moieties were observed to function as coenzymes in the oxidation of malate catalyzed by the purified malate dehydrogenase. Coenzyme-competitive inhibition of the malate dehydrogenase was observed with five adenosine derivatives and six structural analogs of NAD. Of the NAD analogs studied as inhibitors, 3-pyridylcarbinol adenine dinucleotide was the most effective (K(i) = 18 microM). Although inhibition of growth of H. parasuis by this analog was observed, it was less effective (K(i) = 136 microM) than the inhibition of the purified dehydrogenase.
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PMID:Purification and kinetic characterization of Haemophilus parasuis malate dehydrogenase. 924 95


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