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Berlin Brandenburg

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  • 1
    In: The Journal of Bacteriology, 2001, Vol. 183(19), p.5756
    Description: DNA sequence and Southern blot analyses were used to determine the genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire waaQ gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an argininosuccinate synthase homolog, and a change in the 3' sequence of the lgtF gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.
    Keywords: Mutation ; Haemophilus Ducreyi -- Drug Effects ; Lipopolysaccharides -- Metabolism ; Pyocins -- Pharmacology;
    ISSN: 0021-9193
    ISSN: 00219193
    E-ISSN: 10985530
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  • 2
    In: Infection and Immunity, 2007, Vol. 75(1), p.113
    Description: Haemophilus ducreyi is a gram-negative bacterium that is the causative agent of chancroid. Strain 35000HP has been well characterized and is representative of the majority of H. ducreyi strains. Strain 35000HP produces a lipooligosaccharide (LOS) that contains D-glycero-D-manno-heptose in the main oligosaccharide chain extension; the lbgB gene has been shown to encode the DD-heptosyltransferase. The lbgB gene is found in a gene cluster together with the lbgA gene, which encodes for the galactosyltransferase I. These two genes are flanked by two housekeeping genes, rpmE and xthA, encoding the ribosomal protein L31 and the exonuclease III, respectively. Recently, a second group of H. ducreyi strains have been identified. Strain 33921, a representative of the class II strains, produces an LOS that lacks DD-heptose in the oligosaccharide portion of its LOS. To better understand the biosynthesis of the DD-heptose-deficient 33921 LOS, we cloned and sequenced the corresponding lbgAB genomic region from strain 33921. Similar to strain 35000HP, the 33921 genome contains xthA and rpmE. However, between these two genes we identified genes encoding two putative glycosyltransferases that were not highly homologous to the 35000HP lbgAB genes. In this study, we demonstrate that the product of one of these genes encodes a galactosyltransferase. In addition, dot blot hybridization determined that 3 of 35 strains tested had the atypical transferases present, as did 4 strains characterized as class II strains by other criterion. These data indicate that the lbgAB genes can serve as one indicator of the classification of H. ducreyi strains.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 3
    In: Infection and Immunity, 2005, Vol. 73(10), p.6727
    Description: Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.
    Keywords: Bacterial Proteins -- Genetics ; Haemophilus Ducreyi -- Metabolism ; N-Acetylneuraminic Acid -- Metabolism ; Organic Anion Transporters -- Genetics ; Symporters -- Genetics;
    ISSN: 0019-9567
    ISSN: 00199567
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  • 4
    In: Infection and Immunity, 2000, Vol. 68(6), p.3352
    Description: To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 5
    In: Infection and Immunity, 2001, Vol. 69(6), p.4180
    Description: The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.
    Keywords: Mutation ; Chancroid -- Physiopathology ; Glucosyltransferases -- Metabolism ; Haemophilus Ducreyi -- Pathogenicity ; Lipopolysaccharides -- Metabolism;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 6
    In: Infection and Immunity, 2002, Vol. 70(6), p.2853
    Description: All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1--〉4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1--〉6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.
    Keywords: Medicine ; Biology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 7
    Language: English
    In: Molecular microbiology, 2007, Vol.66(1), pp.26-39
    Description: Virulence of nontypeable Haemophilus influenzae (NTHi) is dependent on the decoration of lipooligosaccharide with sialic acid. This sugar must be derived from the host, as NTHi cannot synthesize sialic acids. NTHi can also use sialic acid as a carbon source. The genes encoding the sialic acid transporter and the genes encoding the catabolic activities are localized to two divergently transcribed operons, the siaPT operon and the nan operon respectively. In this study, we identified SiaR as a repressor of sialic acid transport and catabolism in NTHi. Inactivation of siaR resulted in the unregulated expression of the genes in both operons. Unregulated catabolism of sialic acid in the siaR mutant resulted in the reduction of surface sialylation and an increase in serum sensitivity. In addition to SiaR-mediated repression, CRP, the cAMP receptor protein, was shown to activate expression of the siaPT operon but not the nan operon. We describe a model in which SiaR and CRP work to modulate intracellular sialic acid levels. Our results demonstrate the importance of SiaR-mediated regulation to balance the requirement of surface sialylation and the toxic accumulation of intracellular sialic acid. ; Includes references ; p. 26-39.
    Keywords: Cyclic Adenosine Monophosphate;
    ISSN: 0950-382X
    E-ISSN: 13652958
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