The structural heterogeneity of the lipooligosaccharide (LOS) expressed bypathogenic non-typeable Haemophilus influenzae strain NTHi 9274

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsi ble for otitis media in children and of pneumonitis in adults with depresse d resistance. NTHi is acapsular and, therefore, capsular polysaccharide-bas ed vaccines are ineffective for preventing infections by this pathogen, Rec ently it was found that a detoxified lipooligosaccharide (LOS) conjugate fr om NTHi 9274 induced bactericidal antibodies effective against a large numb er of NTHi isolates, and conferred protection against NTHi 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 report the ch emical characterization of the LOS from NTHi 9274 LOS, NTHi is capable of e xpressing a heterogenous population of LOS exhibited by multiple oligosacch aride (OS) epitopes, OSs released from the LOS of NTHi 9274 by mild acid hy drolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage a nalysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardme nt mass spectrometry (FAB-MS), matrix-assisted laser desorption time of fli ght mass spectrometry (MALDITOF-MS), and tandem MS/MS. At least 17 differen t OS molecules were observed. These contained variable glycosyl residues, p hosphate (P), and phosphoethanolamine (PEA) substituents, These molecules c ontained either three, four, or five hexoses, and all contained four heptos yl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep, Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (H F) reduced the number of molecules to about to seven; Hex(1-7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kd o(1) were the major constituents, Thus, this NTHi LOS preparation is very h eterogeneous, and contains structures different from those previously publi shed for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl lin kage data suggest that the LOS oligosaccharides have the following structur es: [GRAPHICS] where Hex is either a Glc or Gal residue.