ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM.IMPORTANCENontypeableHaemophilus influenzae(NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such asNeisseria meningitidis,Moraxella catarrhalis, andBordetella pertussis.

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease‐associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [β‐d‐GalpNAc‐(1→3)‐α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp] and its truncated versions globoside [α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp] and lactose [β‐d‐Galp‐(1→4)‐β‐d‐Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an α‐d‐Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner‐core moiety l‐α‐d‐Hepp‐(1→2)‐[PEtn→6]‐l‐α‐d‐Hepp‐(1→3)‐l‐α‐d‐Hepp‐(1→5)‐[PPEtn→4]‐α‐Kdo‐(2→6)‐lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI‐MS on O‐deacylated LPS and core oligosaccharide material, as well as electrospray ionization‐multiple‐step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis‐ESI‐MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O‐linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.

Overall, the decreased maximal fat oxidation was probably due to lower exogenous plasma fatty acid availability and the muscle adaptation pattern indicates an increased glucose transport capacity and an increased muscle lipolysis capacity supporting an increased contribution of exogenous glucose and endogenous fat during exercise.

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Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. A characteristic feature of H. influenzae LPS is the extensive intra- and inter-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behaviour of the bacterium. The chemical composition of non-typeable Haemophilus influenzae (NTHi) LPS is highly diverse. It contains a number of different monosaccharides (Neu5Ac, L-glycero-D-manno heptose, D-glycero-D-manno heptose, Kdo, D-Glc, D-Gal, D-GlcNAc, D-GalNAc) and non-carbohydrate substituents. Prominent non-carbohydrate components are O-acetyl groups, glycine and phosphates. We now know that sialic acid (N-acetylneuraminic acid or Neu5Ac) and certain oligosaccharide extensions are important in the pathogenesis of NTHi; however, the biological implications for many of the various features are still unknown. Electrospray ionization mass spectrometry in combination with separation techniques like CE and HPLC is an indispensable tool in profiling glycoform populations in heterogeneous LPS samples. Mass spectrometry is characterized by its extreme sensitivity. Trace amounts of glycoforms expressing important virulence determinants can be detected and characterized on minute amounts of material. The present review focuses on LPS structures and mass spectrometric methods which enable us to profile these in complex mixtures.