19 research outputs found
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
Phosphocholine-Modified Lipooligosaccharides of Haemophilus influenzae Inhibit ATP-Induced IL-1beta 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-1beta. 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-1beta release by A549 and Calu-3 cells via nicotinic acetylcholine receptors containing subunits alpha7, alpha9, and/or alpha10. 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
Linking Mass Spectrometry and Slab-Polyacrylamide Gel Electrophoresis by Passive Elution of Lipopolysaccharides from Reverse-Stained Gels: Analysis of Gel-Purified Lipopolysaccharides from Haemophilus
Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176
NRC publication: Ye
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-MSn on dephosphorylated and methylated OS samples. The most abundant glycoform contained a disaccharide chain: PCho\u21927)-D-\u3b1-D-Hepp-(1\u21926)-\u3b2-D-Glcp linked to HepI from the common structural element of H. influenzae LPS: L-\u3b1-D-HepIIIp-(1\u21922)-[PEtn\u21926]-L-\u3b1-D-HepIIp-(1\u21923)-L-\u3b1-D-HepIp-(1\u21925)-[PPEtn\u21924]-\u3b1-Kdop-(2\u21926)-lipid A. Phosphocholine (PCho) was found at two positions in the LPS glycoforms; PCho substituted the 6-position of \u3b2-D-Glcp attached to HepIII and was also located at a novel position linked to D-\u3b1-D-Hepp; this latter position was determined by structural analysis of LPS from a 1158lpsA mutant strain. Additionally, HPLC-ESI-MSn 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.Peer reviewed: YesNRC publication: Ye
Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O-acetyl groups
Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5'-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis
A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae : A survey of 24 non-typeable H. influenzae strains
NRC publication: Ye