Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1ß Release by Pulmonary Epithelial Cells.

Phosphocholine-modified bacterial cell wall components are virulence factors enabling immune evasion and permanent colonization of the mammalian host, by mechanisms that are poorly understood. Recently, we demonstrated that free phosphocholine (PC) and PC-modified lipooligosaccharides (PC-LOS) from Haemophilus influenzae, an opportunistic pathogen of the upper and lower airways, function as unconventional nicotinic agonists and efficiently inhibit the ATP-induced release of monocytic IL-1ß. We hypothesize that H. influenzae PC-LOS exert similar effects on pulmonary epithelial cells and on the complex lung tissue. The human lung carcinoma-derived epithelial cell lines A549 and Calu-3 were primed with lipopolysaccharide from Escherichia coli followed by stimulation with ATP in the presence or absence of PC or PC-LOS or LOS devoid of PC. The involvement of nicotinic acetylcholine receptors was tested using specific antagonists. We demonstrate that PC and PC-LOS efficiently inhibit ATP-mediated IL-1ß release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Primed precision-cut lung slices behaved similarly. We conclude that H. influenzae hijacked an endogenous anti-inflammatory cholinergic control mechanism of the lung to evade innate immune responses of the host. These findings may pave the way towards a host-centered antibiotic treatment of chronic airway infections with H. influenzae.

Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1ß Release.

IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

Comparative genomics of unintrogressed Campylobacter coli clades 2 and 3.

BACKGROUND: Campylobacter jejuni and C. coli share a multitude of risk factors associated with human gastrointestinal disease, yet their phylogeny differs significantly. C. jejuni is scattered into several lineages, with no apparent linkage, whereas C. coli clusters into three distinct phylogenetic groups (clades) of which clade 1 has shown extensive genome-wide introgression with C. jejuni, yet the other two clades (2 and 3) have less than 2% of C. jejuni ancestry. We characterized a C. coli strain (76339) with four novel multilocus sequence type alleles (ST-5088) and having the capability to express gamma-glutamyltranspeptidase (GGT); an accessory feature in C. jejuni. Our aim was to further characterize unintrogressed C. coli clades 2 and 3, using comparative genomics and with additional genome sequences available, to investigate the impact of horizontal gene transfer in shaping the accessory and core gene pools in unintrogressed C. coli. RESULTS: Here, we present the first fully closed C. coli clade 3 genome (76339). The phylogenomic analysis of strain 76339, revealed that it belonged to clade 3 of unintrogressed C. coli. A more extensive respiratory metabolism among unintrogressed C. coli strains was found compared to introgressed C. coli (clade 1). We also identified other genes, such as serine proteases and an active sialyltransferase in the lipooligosaccharide locus, not present in C. coli clade 1 and we further propose a unique scenario for the evolution of Campylobacter ggt. CONCLUSIONS: We propose new insights into the evolution of the accessory genome of C. coli clade 3 and C. jejuni. Also, in silico analysis of the gene content revealed that C. coli clades 2 and 3 have genes associated with infection, suggesting they are a potent human pathogen, and may currently be underreported in human infections due to niche separation.

Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms.

Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host-bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component.

A novel branching pattern in the lipopolysaccharide expressed by non-typeable Haemophilus influenzae strain 1232.

We report the novel branching pattern in lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strain 1232. The strain expressed the ß-d-Glcp-(1→4)-[α-d-Galp-(1→4)-ß-d-Galp-(1→7)]-d-α-d-Hepp-(1→6)-ß-d-Glcp chain linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety of NTHi LPS: l-α-d-HepIIIp-(1→2)-[PEtn→6]-l-α-d-HepIIp-(1→3)-l-α-d-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. The structure has been elucidated using NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MS(n)) on O-deacylated LPS and core oligosaccharide (OS) materials, as well as HPLC-ESI-MS(n) on permethylated, dephosphorylated OS. It was also found that a tetrasaccharide unit bearing sialic acid [α-Neu5Ac-(2→3)-ß-d-Galp-(1→4)-ß-d-GlcNAcp-(1→3)-ß-d-Galp-(1→] could substitute O-4 of the ß-d-Glcp linked to HepI. In addition, the distal heptose (HepIII) was substituted by PCho→6-ß-d-Galp-(1→ at the O-2 position.

The structural diversity of lipopolysaccharide expressed by non-typeable Haemophilus influenzae strains 1158 and 1159.

A heterogeneous population of glycoforms expressed by NTHi strains 1158 and 1159 has been elucidated using NMR spectroscopy and capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) materials, as well as HPLC-ESI-MS(n) on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho→7)-D-α-D-Hepp-(1→6)-ß-D-Glcp linked to HepI from the common structural element of H. influenzae LPS: L-α-D-HepIIIp-(1→2)-[PEtn→6]-L-α-D-HepIIp-(1→3)-L-α-D-HepIp-(1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of ß-D-Glcp attached to HepIII and was also located at a novel position linked to D-α-D-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MS(n) experiments indicated glycoforms that have chain elongation from HepII, this was found only in glycoforms, which lack the additional heptose in the outer core region. Structural details of these glycoforms were confirmed by analyses of LPS from a 1158losB2 mutant strain; the losB2 gene is required for addition of the D,D-Hep to the outer core region in strain 1158.

Identification and characterization of a lipopolysaccharide α,2,3-sialyltransferase from the human pathogen Helicobacter bizzozeronii.

Terminal sialic acid in the lipopolysaccharides (LPSs) of mucosal pathogens is an important virulence factor. Here we report the characterization of a Helicobacter sialyltransferase involved in the biosynthesis of sialylated LPS in Helicobacter bizzozeronii, the only non-pylori gastric Helicobacter species isolated from humans thus far. Starting from the genome sequences of canine and human strains, we identified potential sialyltransferases downstream of three genes involved in the biosynthesis of N-acetylneuraminic acid. One of these candidates showed monofunctional α,2,3-sialyltransferase activity with a preference for N-acetyllactosamine as a substrate. The LPSs from different strains were shown by SDS-PAGE and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to contain sialic acid after neuraminidase treatment. The expression of this sialyltransferase and sialyl-LPS appeared to be a phase-variable characteristic common to both human and canine H. bizzozeronii strains. The sialylation site of the LPSs of two H. bizzozeronii strains was determined to be NeuAc-Hex-HexNAc, suggesting terminal 3'-sialyl-LacNAc. Moreover, serological typing revealed the possible presence of sialyl-Lewis X in two additional strains, indicating that H. bizzozeronii could also mimic the surface glycans of mammalian cells. The expression of sialyl-glycans may influence the adaptation process of H. bizzozeronii during the host jump from dogs to humans.

Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20.

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20. Using NMR and MS techniques on LPS, oligosaccharide samples and lipid A, the structures for O-antigen, core oligosaccharide and lipid A could be established. It was found that the biological repeating unit of the O-antigen is →4)-α-D-GalpNAc-(1→P→6)-ß-D-Glcp-(1→3)-α-D-FucpNAc4N-(1→, in which D-FucpNAc4N is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. This sugar is in ß-configuration when linked to O-4 of the glucose residue of ß-D-Galp-(1→2)-L-α-D-Hepp-(1→2)-[PEtn→6]-L-α-D-Hepp-(1→3)-[ß-D-Glcp-(1→4)]-L-α-D-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. LPS from a wbaP mutant of H. parainfluenzae strain 20 did not contain an O-antigen, consistent with the wbaP gene product being required for expression of O-antigen in fully extended LPS.

Expression of a new disialyllacto structure in the lipopolysaccharide of non-typeable Haemophilus influenzae.

The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [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] with PCho→6)-ß-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [ß-d-GalpNAc-(1→3)-α-d-Galp-(1→4)-ß-d-Galp-(1→4)-ß-d-Glcp-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-ß-d-Galp-(1→4)-ß-d-Glcp-(1→] and lactose [ß-d-Galp-(1→4)-ß-d-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-ß-d-Gal-(1→4)-ß-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.

The role of lic2B in lipopolysaccharide biosynthesis in Haemophilus influenzae strain Eagan.

Lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae involves genes from the lic2 locus that are required for chain extension from the middle heptose (HepII) of the conserved triheptosyl inner-core moiety. Lic2C initiates the process by attaching the first glucose to HepII, but the gene encoding for the enzyme adding the next ß-D-Glcp- is uncharacterized. Lic2B is the candidate glucosyltransferase; however, in previous investigations, mutation of lic2B resulted in no hexose extension from HepII, likely due to a polar effect on the lic2C gene. In this study we complemented a lic2B knock-out mutant of H. influenzae strain Eagan with a functional lic2C gene and investigated its LPS by mass spectrometry and 2D NMR spectroscopy. Lic2B was found to encode a glucosyltransferase responsible for the linkage of ß-D-Glcp-(1→4)-α-D-Glcp-(1→ extending from O-3 of the central heptose 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.

Detailed structural features of lipopolysaccharide glycoforms in nontypeable Haemophilus influenzae strain 2019.

We have investigated the structure of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae (NTHi) strain 2019, a prototype strain that is used for studies of NTHi biology and disease. Analysis of LPS from wild type and lex2B, lpt3 and pgm mutant strains using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MS(n) on permethylated dephosphorylated OS, confirmed the previously established structure in which lactose is linked to the proximal heptose (HepI) of the conserved 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. Importantly, our data provide further structural detail whereby extensions from the middle heptose (HepII) are now characterized as ß-D-Galp-(1→4)-ß-D-Glcp-(1→4)-α-D-Glcp-(1→3 and truncated versions thereof. PEtn substitutes O-3 of the distal heptose (HepIII) of the inner-core moiety. This PEtn substituent was absent in the lpt3 mutant indicating that Lpt3 is the transferase required to add PEtn to the distal heptose. Interestingly, in the lex2B mutant strain HepIII was found to be substituted at O-2 by ß-D-Glcp which, in turn, can be further extended. Contrary to previous findings, LPS of the pgm mutant strain contained minor glycoforms having ß-D-Glcp linked to O-4 of HepI and also glycoforms with an additional PEtn which could be assigned to HepIII. Acetate groups and one glycine residue further substitute HepIII in NTHi 2019.

The T-helper cell type 1 immune response to gram-negative bacterial infections is impaired in COPD.

RATIONALE: The increased susceptibility to bacterial infections in chronic obstructive pulmonary disease (COPD) is critical for exacerbations. Toll-like receptor-4 (TLR4) detects bacteria via LPS and induces IFN-γ-based immune responses. The direct responsiveness of Th1 lymphocytes to LPS is disputed because they lack surface expression of the TLR4 coreceptor CD14. OBJECTIVES: We hypothesized that the Th1-mediated adaptive immune response to bacterial infections is impaired in COPD. METHODS: LPS-induced TLR4 expression and IFN-γ release in and from ex vivo-generated Th1 cells was compared among nonsmokers (n = 14), smokers without COPD (n = 13), and smokers with COPD (n = 25) via quantitative reverse transcription polymerase chain reaction, Western blot, and ELISA. TLR4 transfection experiments were performed to functionally link receptor to IFN-γ dysregulation in COPD. MEASUREMENTS AND MAIN RESULTS: Short-chain LPS from Salmonella species and nontypeable Haemophilus influenzae and nontypeable Haemophilus influenzae whole-cell extract all induced TLR4 expression via TLR4/MyD88/IRAK/mitogen-activated protein-kinase signaling and IFN-γ release via TLR4/TRIF/IKKε/TBK1 signaling in Th1 cells of nonsmokers. These effects were all impaired in smokers with and without COPD. The LPS responses were partially dependent on soluble CD14 and correlated positively to lung-function parameters but negatively to cigarette smoking (pack-years). Endogenous MyD88/IRAK signaling antagonists were up-regulated in Th1 cells of smokers and COPD, and TLR4 overexpression in Th1 cells of COPD restored LPS-dependent IFN-γ release. CONCLUSIONS: Th1 cells directly respond to short-chain LPS. Cigarette smoking suppresses Th1-mediated immune responses to gram-negative bacterial infections by interfering with MyD88/IRAK signaling thereby reducing LPS-induced TLR4 expression. This can explain the increased susceptibility to bacterial infections in COPD. Targeting TLR signaling might be useful to reduce exacerbation rates.

Genes required for the synthesis of heptose-containing oligosaccharide outer core extensions in Haemophilus influenzae lipopolysaccharide.

Heptose-containing oligosaccharides (OSs) are found in the outer core of the lipopolysaccharide (LPS) of a subset of non-typable Haemophilus influenzae (NTHi) strains. Candidate genes for the addition of either l-glycero-d-manno-heptose (ld-Hep) or d-glycero-d-manno-heptose (dd-Hep) and subsequent hexose sugars to these OSs have been identified from the recently completed genome sequences available for NTHi strains. losA1/losB1 and losA2/losB2 are two sets of related genes in which losA has homology to genes encoding glycosyltransferases and losB to genes encoding heptosyltransferases. Each set of genes is variably present across NTHi strains and is located in a region of the genome with an alternative gene organization between strains that contributes to LPS heterogeneity. Dependent upon the strain background, the LPS phenotype, structure and serum resistance of strains mutated in these genes were altered when compared with the relevant parent strain. Our studies confirm that losB1 and losB2 usually encode dd-heptosyl- and ld-heptosyl transferases, respectively, and that losA1 and losA2 encode glycosyltransferases that play a role in OS extensions of NTHi LPS.

Profiling LPS glycoforms of non-typeable Haemophilus influenzae by multiple-stage tandem mass spectrometry.

Non-typeable (acapsular) Haemophilus influenzae (NTHi) is a major cause of otitis media accounting for 25-30% of all cases of the disease. Lipopolysaccharide (LPS) is an essential and exposed component of the H. influenzae cell wall. A characteristic feature of H. influenzae LPS is the extensive inter-strain and intra-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behavior of the bacterium. However, to characterize LPS structure unambiguously is a major challenge due to the extreme heterogeneity of glycoforms that certain strains express. A powerful tool for obtaining sequence and branching information is multiple-stage tandem ESI-MS (ESI-MS( n )) performed on dephosphorylated and permethylated oligosaccharide material using an ESI-quadrupole ion trap mass spectrometer. In general, permethylation increases the MS response by several orders of magnitude and sequence information is readily obtained since methyl tagging allows the distinction between fragment ions generated by cleavage of a single glycosidic bond and inner fragments resulting from the rupture of two glycosidic linkages. Using this approach we are now able to identify all isomeric glycoforms in very heterogeneous LPS preparations.

A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019.

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-GlcIp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-alpha-Kdop) to which addition of beta-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a beta-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of beta-d-Galp-(1-->4-beta-d-Glcp-(1--> from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMS(n) on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only beta-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a beta-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.

Rapid method for sensitive screening of oligosaccharide epitopes in the lipooligosaccharide from Campylobacter jejuni strains isolated from Guillain-Barré syndrome and Miller Fisher syndrome patients.

Campylobacter jejuni lipooligosaccharide (LOS) can trigger Guillain-Barré syndrome (GBS) due to its similarity to human gangliosides. Rapid and accurate structural elucidation of the LOS glycan of a strain isolated from a GBS patient could help physicians determine the spectrum of anti-ganglioside antibodies likely to be found and therefore provide valuable assistance in establishing an appropriate course of treatment. The ability of implemented mass spectrometry-based approaches in a clinical setting has been limited by the laborious and time-consuming nature of the protocols, typically 3 to 4 days, used to prepare LOS. In order to improve the analytical throughput, microwave-assisted enzymatic digestion was investigated. In this study, the bacterial cells were suspended in 50 microl of 20 mM ammonium acetate buffer containing DNase and RNase and treated by direct microwave irradiation for 3 min. Then, proteinase K was added and the samples were again microwaved. The intact LOS samples were analyzed using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry. The reliability of the rapid, high-throughput technique was demonstrated through analysis of LOS glycans from 73 C. jejuni strains. The structure was elucidated using material from a single colony. The total time for sample preparation and MS analysis is less than 60 min.

Profiling structural elements of short-chain lipopolysaccharide of non-typeable Haemophilus influenzae.

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.

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain R2846.

We here report the lipopolysaccharide (LPS) structures expressed by nontypeable Haemophilus influenzae R2846, a strain whose complete genome sequence has recently been obtained. Results were obtained by using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS (n) on permethylated dephosphorylated OS. A beta- d-Glc p-(1-->4)- d-alpha- d-Hep p-(1-->6)-beta- d-Glc p-(1-->4) unit was found linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety, l-alpha- d-Hep p-(1-->2)-[ PEtn-->6]- l-alpha- d-Hep p-(1-->3)- l-alpha- d-Hep p-(1-->5)-[ PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. The beta- d-Glc p (GlcI) linked to HepI was also branched with oligosaccharide extensions from O-4 and O-6. O-4 of GlcI was substituted with sialyllacto- N-neotetraose [alpha-Neu5Ac-(2-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->] and the related structure [( PEtn-->6)-alpha- d-Gal pNAc-(1-->6)-beta- d-Gal p-(1-->4)-beta- d-Glc pNAc-(1-->3)-beta- d-Gal p-(1-->4)-beta- d-Glc p-(1-->]. The distal heptose (HepIII) was substituted at O-2 by beta- d-Gal. Phosphate, phosphoethanolamine, phosphocholine, acetate, and glycine were found to substitute the core oligosaccharide. Two heptosyltransferase genes, losB1 and losB2, have been identified from the R2846 genome sequence and are candidates to add the noncore heptose to the LPS. Mutant strain R2846 losB1 did not show dd-heptose in the extension from HepI but still contained minor quantities of ld-heptose at the same position, indicating that the losB1 gene is required to add dd-heptose to GlcI. The LPS from strain R2846 losB1/ losB2 expressed no noncore heptose, consistent with losB2 directing the addition of ld-heptose.

Application of capillary electrophoresis mass spectrometry and liquid chromatography multiple-step tandem electrospray mass spectrometry to profile glycoform expression during Haemophilus influenzae pathogenesis in the chinchilla model of experimental otitis media.

Otitis media caused by nontypeable Haemophilus influenzae (NTHi) is a common and recurrent bacterial infection of childhood. The structural variability and diversity of H. influenzae lipopolysaccharide (LPS) glycoforms are known to play a significant role in the commensal and disease-causing behavior of this pathogen. In this study, we determined LPS glycoform populations from NTHi strain 1003 during the course of experimental otitis media in the chinchilla model of infection by mass spectrometric techniques. Building on an established structural model of the major LPS glycoforms expressed by this NTHi strain in vitro (M. Månsson, W. Hood, J. Li, J. C. Richards, E. R. Moxon, and E. K. Schweda, Eur. J. Biochem. 269:808-818, 2002), minor isomeric glycoform populations were determined by liquid chromatography multiple-step tandem electrospray mass spectrometry (LC-ESI-MS(n)). Using capillary electrophoresis ESI-MS (CE-ESI-MS), we determined glycoform profiles for bacteria from direct middle ear fluid (MEF) samples. The LPS glycan profiles were essentially the same when the MEF samples of 7 of 10 animals were passaged on solid medium (chocolate agar). LC-ESI-MS(n) provided a sensitive method for determining the isomeric distribution of LPS glycoforms in MEF and passaged specimens. To investigate changes in LPS glycoform distribution during the course of infection, MEF samples were analyzed at 2, 5, and 9 days postinfection by CE-ESI-MS following minimal passage on chocolate agar. As previously observed, sialic acid-containing glycoforms were detected during the early stages of infection, but a trend toward more-truncated and less-complex LPS glycoforms that lacked sialic acid was found as disease progressed.

Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry.

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.