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Infection and Immunity, June 2000, p. 3352-3361, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Construction and Characterization of Haemophilus
ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression
of Heptosyltransferase III and
1,4-Glucosyltransferase:
Identification of LOS Glycoforms Containing Lactosamine
Repeats
Melanie J.
Filiatrault,1,2
Bradford W.
Gibson,3
Birgit
Schilling,3
Shuhua
Sun,4,5
Robert S.
Munson Jr.,4,5 and
Anthony
A.
Campagnari1,2,6,*
Department of Microbiology,1
Department of Medicine, Division of Infectious
Diseases,6 and Center for Microbial
Pathogenesis,2 University at Buffalo, Buffalo,
New York 14214; Department of Pharmaceutical Chemistry,
University of California, San Francisco, California
94143-04463; and Children's Research
Institute4 and Department of
Molecular Virology, Immunology, and Medical
Genetics,5 Ohio State University, Columbus, Ohio
43205-2696
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.
*
Corresponding author. Mailing address: Department of
Microbiology, University at Buffalo, Biomedical Research Bldg. Rm. 143, 3435 Main St., Buffalo, NY 14214. Phone: (716) 829-2673. Fax: (716)
829-3889. E-mail: AAC{at}acsu.buffalo.edu.
Infection and Immunity, June 2000, p. 3352-3361, Vol. 68, No. 6
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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