NDLI: Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

Content Provider	PubMed Central
Author	Filiatrault, Melanie J. Gibson, Bradford W. Schilling, Birgit Sun, Shuhua Munson, Robert S. Campagnari, Anthony A.
Editor	Burns, D. L.
Copyright Year	2000
Abstract	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.
Starting Page	3352
File Format	PDF
ISSN	10985522
e-ISSN	10985522
Journal	Infection and Immunity
Issue Number	6
Volume Number	68
Language	English
Publisher	American Society for Microbiology
Publisher Date	2000-06-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Parasitology Immunology Microbiology

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Haemophilus ducreyi Lipooligosaccharide Mutant Defective in Expression of β-1,4-Glucosyltransferase Is Virulent in Humans

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

Use of pyocin to select a Haemophilus ducreyi variant defective in lipooligosaccharide biosynthesis.

The lbgAB Gene Cluster of Haemophilus ducreyi Encodes a β-1,4-Galactosyltransferase and an α-1,6-dd-Heptosyltransferase Involved in Lipooligosaccharide Biosynthesis

Identification of Genes Involved in the Expression of Atypical Lipooligosaccharide Structures from a Second Class of Haemophilus ducreyi ▿

Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

Identification and Characterization of the N-Acetylglucosamine Glycosyltransferase Gene of Haemophilus ducreyi

Characterization of the CpxRA Regulon in Haemophilus ducreyi ▿ †

Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

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Haemophilus ducreyi Lipooligosaccharide Mutant Defective in Expression of β-1,4-Glucosyltransferase Is Virulent in Humans

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