CHARACTERIZATION OF A TRANSPOSON-TN916-GENERATED MUTANT OF HAEMOPHILUS-DUCREYI-35000 DEFECTIVE IN LIPOOLIGOSACCHARIDE BIOSYNTHESIS

To define the role of the surface lipooligosaccharide (LOS) of Haemoph ilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. duc reyi 35000 defective in expression of the murine monoclonal antibody ( MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic scr eening, One mutant, designated 1381, has an LOS which lacks the MAb 3F 11 epitope and migrates with an increased mobility on sodium dodecyl s ulfate-polyacrylamide gel electrophoresis, The gene disrupted by the T n916 element in strain 1381 was identified by cloning the sequences fl anking the Tn916 element, The sequences were then used to probe a lamb da DASHII genomic library, In strain 1381, Tn916 interrupts a gene whi ch encodes an open reading frame (ORF) with an M-r of 40,246. This ORF has homology to the product of the rfaK gene of Escherichia coli. The major LOS glycoform produced by strain 1381 was analyzed by using a c ombination of mass spectrometry, linkage and composition analysis, and H-1 nuclear magnetic resonance spectroscopy, The major LOS species wa s found to terminate in a single glucose attached to the heptose (L-gl ycero-D-manno-heptose, or Hep) trisaccharide core, In the wild-type st rain 35000, glucose serves as the acceptor for the addition of the D-g lycero-D-manno-heptose (or DDHep). which extends to form the mature br anch of the H. ducreyi LOS, This mature oligosaccharide is in turn par tially capped by the addition of sialic acid (NeuAc), i.e., NeuAc2 alp ha-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4DDHep alpha 1-->6Gl c beta 1 (W. Melaugh et al., Biochemistry 33:13070-13078, 1994), Since this LOS terminates prior to the addition of the branch DD-heptose, t his gene is likely to encode the D-glycero-D-manno-heptosyltransferase . Strain 1381 exhibits a significant reduction in adherence to and inv asion of primary human keratinocytes. This defect was complemented by the cloned heptosyltransferase gene, indicating that the terminal port ion of the LOS oligosaccharide plays an important role in adherence to human keratinocytes.