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.