Abstract
The first preliminary structure of a surface lipooligosaccharide from Haemophilus ducreyi has been determined. The major oligosaccharide was released by mild acid hydrolysis and analyzed by liquid secondary ion and tandem mass spectrometry. The mass spectral data combined with composition and methylation analysis yielded the most probable structure; Gal1----4GlcNAc1----3Gal1----4Hep1----6Glc1----( Hep1----2Hep1----)3,4Hep1---- KDO, where the reducing terminal 3-deoxy-D-manno-octulosonic acid (or KDO) exists in an anhydro form. This anhydro species results from the elimination of a phosphate from C-4 of KDO during mild acid hydrolysis. The core heptose trisaccharide consists of L-glycero-D-manno-heptose, but analysis of the peracetylated sugars indicated that the 1,4-linked heptose is likely D-glycero-D-manno-heptose. The monoclonal antibody 3F11 generated against Neisseria gonorrhoeae also binds to this lipooligosaccharide and suggests that the terminal trisaccharide is Gal beta 1----4GlcNAc beta 1----3Gal beta 1----, an epitope found in the glycose moiety of the human erythrocyte glycosphingolipid lactoneotetraglycosylceramide. Mass spectrometric and composition analysis of the lipid A moiety shows that it is similar to the lipid A of Haemophilus influenzae strain I-69 Rd-/b+ proposed by Helander et al. (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). Electrospray mass spectrometric analysis of the intact O-deacylated lipooligosaccharides gave an average Mr of 2710, and supported an overall structure consisting of the above nonasaccharide linked directly to a diphosphorylated lipid A moiety through the single KDO which is phosphorylated. This structure should provide a framework to investigate the roles of lipooligosaccharides in the host immunochemical response and pathology of H. ducreyi infection, a leading cause of genital ulcer disease.
Highlights
Partial Characterization of the Major Lipooligosaccharide from a Strain of Haemophilus ducreyi, the Causative Agent of Chancroid, a Genital Ulcer Disease
The first preliminary structureof a surface lipooli- Haemophilus ducreyi is a Gram-negative human mucosal gosaccharide from Haemophilus ducreyi has been de- pathogen that is thperinciple cause of genital ulcer diseasein termined
H.ducreyi infection mild acid hydrolysis and analyzedby liquid secondary is endemic in large parts of Africa and Asia, and it harsecently ion and tandem mass spectrometry
Summary
Acetonitrile, were added to 2 volumesof the hydrolysis mixture and vortexed This water, and methanol were obtained from Burdick and Jackson (Mu- mixture was centrifuged a t 5,000 X g for 15 min and thelower organic skegon, MI). The dried organisms were raphy (TLC) of the resulting lipid A fraction was performed using suspended in a mixture of hot water/phenol which was kept at 65 "C 250-pm silica gel LK5 plates (Whatman) as described by Johnson et for 10-15 min. The sample was cooled to -20 "C and chilled mixture was injected that containedequimolar amounts of the followacetone added dropwise to precipitate the 0-deacylated LOS which ing sugars: terminal Gal, terminal Hep,1,2-linked Gal, 1,2-linked was centrifuged a t 12,000 X g for 20 min. The precipitated 0-deacylated LOS was resuspended in oligosaccharides were directly analyzed by liquid secondary ion mass 500 pl of water and lyophilized.
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