An Alternate Pattern for Globoside Oligosaccharide Expression in Haemophilus influenzae Lipopolysaccharide: Structural Diversity in Nontypeable Strain 1124 Håkan H. Yildirim, ‡ Jianjun Li, § James C. Richards, § Derek W. Hood, | E. Richard Moxon, | and Elke K. H. Schweda* ,‡ Clinical Research Centre, Karolinska Institutet and UniVersity College of South Stockholm, NOVUM, S-141 86 Huddinge, Sweden, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A OR6, and Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, U.K. ReceiVed December 1, 2004; ReVised Manuscript ReceiVed January 25, 2005 ABSTRACT: Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [-D-GalpNAc-(1f3)-R-D-Galp-(1f4)--D-Galp-(1f4)--D-Glcp] and its truncated versions globoside [R-D-Galp-(1f4)--D-Galp-(1f4)--D-Glcp] and lactose [-D-Galp-(1f4)--D-Glcp] linked to the terminal heptose (HepIII) of the triheptosyl inner-core moiety L-R-D-Hepp-(1f2)-[PEAf6]-L-R-D-Hepp-(1f3)- L-R-D-Hepp-(1f5)-[PPEAf4]-R-Kdo-(2f6)-lipid A. We report here structural studies of LPS from nontypeable H. influenzae strain 1124 expressing these motifs linked to both the proximal heptose (HepI) and HepIII at the same time. This novel finding was obtained by structural studies of LPS using NMR techniques and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS n on permethylated dephosphorylated OS. The use of defined mutants allowed us to confirm structures unambiguously and understand better the biosynthesis of each of the globotetraose units. We found that lgtC is involved in the expression of R-D-Galp-(1f4)- -D-Galp in both extensions, whereas lic2A directs only the expression of -D-Galp-(1f4)--D-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MS/MS. A common sialylated structure in H. influenzae LPS is sialyllactose linked to HepIII. This structure exists in strain 1124. However, results for the lpsA mutant indicate that sialyllactose extends from HepI as well, a molecular environment for sialyllactose in H. influenzae that has not been reported previously. In addition, the LPS was found to carry phosphorylcholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII. Haemophilus influenzae is an important cause of human disease worldwide and exists in encapsulated (type a-f) and unencapsulated (nontypeable) forms. Type b capsular strains are associated with invasive bacteraemic diseases, including meningitis, epiglottis, cellulitits, and pneumonia, while acapsular or nontypeable strains of H. influenzae (NTHi) 1 are primary pathogens in otitis media and both acute and chronic lower respiratory tract infections (1). The potential of H. influenzae to cause disease depends on its surface- expressed carbohydrate antigens, capsular polysaccharide (2), and lipopolysaccharide (LPS) (3). LPS is an essential and characteristic surface component of H. influenzae. This bacterium has been found to express short-chain LPS, lacking O-specific polysaccharide chains and often termed lipooligosaccharide (LOS). Extensive structural studies of LPS from H. influenzae by us and others have led to the identification of a conserved glucose- substituted triheptosyl inner-core moiety L-R-D-Hepp-(1f2)- [PEAf6]-L-R-D-Hepp-(1f3)-[-D-Glcp-(1-4)]-L-R-D- Hepp linked to lipid A via 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) 4-phosphate. This inner-core unit provides the template for attachment of oligosaccharide and noncarbo- hydrate substituents (4-23). LPS of H. influenzae can mimic host glycolipids and has a propensity for reversible switching of expression of saccharide and noncarbohydrate epitopes (phase variation), leading to considerable intrastrain variation in structure. Phase variation is thought to provide an adaptive mechanism which is advantageous for survival of bacteria confronted by the differing microenvironments and immune responses of the host (24). Terminal structures mimicking the globoside series of mammalian glycolipids have been identified. These include galabiose [R-D-Galp-(1f4)--D- Galp-(1f], lactose [-D-Galp-(1f4)--D-Glcp-(1f], sia- lyllactose [R-Neu5Ac-(2f3)--D-Galp-(1f4)--D-Glcp- (1f], and globotetraose [-D-GalpNAc-(1f3)-R-D-Galp- * To whom correspondence should be addressed: University College of South Stockholm, Clinical Research Center, NOVUM, S-141 86 Huddinge, Sweden. Telephone: + 46 8 585 838 23. Fax: + 46 8 585 838 20. E-mail: elke.schweda@kfc.ki.se. ‡ Karolinska Institutet and University College of South Stockholm. § National Research Council of Canada. | John Radcliffe Hospital. 1 Abbreviations: CE, capillary electrophoresis; Kdo, 3-deoxy-D- manno-oct-2-ulosonic acid; AnKdo-ol, reduced anhydro-Kdo; Hep, heptose; L,D-Hep, L-glycero-D-manno-heptose; Hex, hexose; LPS-OH, O-deacylated lipopolysaccharide; lipid A-OH, O-deacylated lipid A; LPS, lipopolysaccharide; MS n , multiple-step tandem mass spectrometry; Neu5Ac, N-acetylneuraminic acid; NTHi, nontypeable H. influenzae; OS, oligosaccharide; PCho, phosphocholine; PEA, phosphoethanola- mine; PPEA, pyrophosphoethanolamine; Ac, acetate; Gly, glycine. 10.1021/bi047480h CCC: $30.25 © xxxx American Chemical Society PAGE EST: 17.2 Published on Web 00/00/0000