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Glycobiology, 1999, Vol. 9, No. 12 1371-1380
© 1999 Oxford University Press

The structural heterogeneityof the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274

M.Mahbubur Rahman, Xin-Xing Gu2, Chao-Ming Tsai3, V.S.Kumar Kolli and Russell W.Carlsona

Complex Carbohydrate Research Center, The University ofGeorgia, 220 Riverbend Road, Athens, GA, USA, 2Laboratoryof Immunology, National Institute on Deafness and Other CommunicationDisorders, S. Research Court, Rockville, MD 20850, USA,and 3Center for Biologics Evaluationand Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda,MD 20892, USA

Nontypeable Haemophilus influenzae (NTHi)is an important pathogen responsible for otitis media in childrenand of pneumonitis in adults with depressed resistance. NTHi is acapsularand, therefore, capsular polysaccharide-based vaccines are ineffectivefor preventing infections by this pathogen. Recently it was foundthat a detoxified lipooligo­saccharide (LOS) conjugatefrom NTHi 9274 induced bactericidal antibodies effective againsta large number of NTHi isolates, and conferred protection againstNTHi otitis media in chinchillas (X.-X.Gu et al.,1996, Infect. Immun., 64, 4047–4053;X.-X.Gu et al., 1997., Infect. Immun., 65, 4488–4493). In this paper we reportthe chemical character­ization of the LOS from NTHi 9274LOS. NTHi is capable of expressing a heterogenous population ofLOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released fromthe LOS of NTHi 9274 by mild acid hydrolysis were purified usingBio-Gel P4 gel permeation chromatography. The OSs were characterizedby glycosyl composition analysis, glycosyl linkage analysis, nuclearmagnetic resonance spectroscopy (NMR), fast atom bombardment massspectro­metry (FAB-MS), matrix-assisted laser desorptiontime of flight mass spectro­metry (MALDITOF-MS), and tandem MS/MS.At least 17 different OS molecules were observed. These containedvariable glycosyl residues, phosphate (P), and phospho­ethanolamine(PEA) substituents. These molecules contained either three, four,or five hexoses, and all contained four heptosyl residues. The fourheptosyl residues consisted of one D,D-Hep andthree L,D-Hep. Dephosphorylation of the OSs withaqueous 48% hydrofluoric acid (HF) reduced the number ofmolecules to about to seven; Hex1-7Hep4Kdo1.Of these seven, Hex2Hep4Kdo1, Hex3Hep4Kdo1,and Hex4Hep4Kdo1 were the majorconstituents. Thus, this NTHi LOS preparation is very heterogeneous,and contains structures different from those previously publishedfor Haemophilus influenzae. The tandem MS/MSanalysis and glycosyl linkage data suggest that the LOS oligosaccharides havethe following structures:

Hex-(1->4)-[Glc]0-4-(1->4)-D,D-Hep-(1->6)-Glc-(1->4)-L,D-Hep-(1->5)-Kdo

3

{uparrow}

1

L,D-Hep

2

{uparrow}

1

(Hex)0–1-(1->2)-L,D-Hep

where Hex is either a Glc or Gal residue.

a Towhom correspondence should be addressed at: Complex Carbohydrate ResearchCenter, The University of Georgia, 220 Riverbend Road, Athens, GA 30602


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