Structural studies of lipopolysaccharides expressed by non-typeable Haemophilus influenzae and Haemophilus parainfluenzae strains

The present thesis describes lipopolysaccharide (LPS) structures expressed by non-typeable Haemophilus influenzae and Haemophilus parainfluenzae strains. LPS is a major surface component of Gram-negative bacteria. Structural studies of LPS are very important for understanding the adaptive mechanisms which help bacteria to survive in the host environment. Non-typeable Haemophilus influenzae (NTHi) is a common human commensal of the nasopharynx. It is also pathogenic and causes both acute and chronic diseases, such as otitis media, sinusitis, pneumonia and bronchitis. H. influenzae expresses rough type LPS (lacking O-antigen), which is implicated as a major virulence factor. 25 NTHi otitis media isolates were selected for structural studies of LPS. These clinical isolates represent the structural diversity of LPS in the natural population. Structural studies of H. influenzae LPS have resulted in a molecular model consisting of a conserved (PEtn)-substituted triheptosyl inner-core moiety (HepI–HepII-HepIII) in which each of the heptose residues can provide a point for elongation by oligosaccharide chains (outer-core region). NTHi strains 1158/1159 and 1232, described in this thesis, were selected from this collection of clinical isolates. These strains express additional D,D-Hep residue in the outer-core region of LPS. Haemophilus parainfluenzae is a part of normal human flora. Previous studies have indicated that H. parainfluenzae expresses LPS structures that are very similar to those expressed by H. influenzae. On the other hand some H. parainfluenzae strains express O-antigen containing LPS. The structures of the O-antigen from H. parainfluenzae strains 20 and 16 are described in this thesis. The structural investigations of LPS of H. influenzae and the comparison with LPS expressed by H. parainfluenzae will increase the knowledge of biological properties of LPS and its role in bacterial virulence

Association between hypogonadism and reproductive tissue steroid-producing cells antibody in men with positive MAR test IgG and diabetes mellitus type 1

BACKGROUND: Autoimmune hypogonadism is frequently taped in men with positive direct mixed agglutination reaction antisperm antibodies IgG test (MAR test IgG). AIMS: Тo assess pathogenetic factor of autoimmune hypogonadism in men with positive MAR test IgG and diabetes mellitus type 1 (DM1). MATERIALS AND METHODS: A retrospective study included 97 patients with positive direct MAR test IgG: 30 men with DM1 and 67 – without DM. Assessment included testosterone level and titer of summary reproductive tissue steroid-producing cells antibody (LCA). Statistically significant differences were p<0,05. RESULTS: 43% of men with DM1 have abnormal LCA titer and it was significantly higher than in patients without DM – 21%. In both groups testosterone level was significantly lower in men with abnormal LCA titer than in patients with normal antibodies titer. Frequency of hypogonadism in men with abnormal LCA titer was significantly higher than in patients with normal antibodies titer also in both groups. There were no significantly differences of MAR test IgG in patients with normal and abnormal LCA titer. CONCLUSIONS: Autoimmune hypogonadism is a common complication in men with DM1 and positive MAR test IgG and it’s strongly associated with high titer of summary reproductive tissue steroid-producing cells antibody

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)-β-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 β-configuration when linked to O-4 of the glucose residue of β-D-Galp-(1→2)-L-α-D-Hepp-(1→2)-[PEtn→6]-L-α-D-Hepp-(1→3)-[β-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

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 \u3b2-D-Glcp-(1\u21924)-[\u3b1-D-Galp-(1\u21924)-\u3b2-D-Galp- (1\u21927)]-D-\u3b1-D-Hepp-(1\u21926)-\u3b2-D-Glcp chain linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety of NTHi LPS: L-\u3b1-D-HepIIIp-(1\u21922)-[PEtn\u21926]-L-\u3b1-D-HepIIp-(1\u21923)- L-\u3b1-DHepIp-( 1\u21925)-[PPEtn\u21924]-\u3b1-Kdop-(2\u21926)-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-MSn) on O-deacylated LPS and core oligosaccharide (OS) materials, as well as HPLC-ESI-MSn on permethylated, dephosphorylated OS. It was also found that a tetrasaccharide unit bearing sialic acid [\u3b1-Neu5Ac-(2\u21923)-\u3b2-D-Galp-(1\u21924)- \u3b2-D-GlcNAcp-(1\u21923)-\u3b2-D-Galp-(1\u2192] could substitute O-4 of the \u3b2-D-Glcp linked to HepI. In addition, the distal heptose (HepIII) was substituted by PCho\u21926-\u3b2-D-Galp-(1\u2192 at the O-2 position. \ua9 2013 Published by Elsevier Ltd.Peer reviewed: YesNRC 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

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

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 Fuc-NAc4N 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 Haemophilus influenzae strain associated with Fisher syndrome expresses a novel disialylated ganglioside mimic: Biochemistry

The non-typeable Haemophilus influenzae strain DH1 was isolated from a 25 year old male patient with Fisher syndrome, a postinfectious autoimmune condition characterized by the presence of anti-GQ1b IgG antibodies that target and initiate damage to peripheral nerves. DH1 was found to display an alphaNeuAc(2-8)alphaNeuAc(2-3)betaGal branch bound to the tetraheptosyl backbone core of its lipooligosaccharide (LOS). The novel sialylation pattern was found to be dependent on the activity of a bifunctional sialyltransferase, Lic3B, which catalyzes the addition of both the terminal and subterminal sialic acid residues. Patient serum IgGs bind to DH1 LOS, and the reactivity is significantly influenced by the presence of sialylated glycoforms. The display by DH1, of a surface glycan that mimics the terminal trisaccharide portion of disialosyl-containing gangliosides, provides strong evidence for its involvement in the development of Fisher syndromeNRC publication: Ye

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors.

Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant) and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC) coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSn). The LC-ESI-MSn of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline) and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC).Mucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from serous and high-grade mucinous carcinomas based on their O-glycan profiles

Duplicate Copies of lic1 Direct the Addition of Multiple Phosphocholine Residues in the Lipopolysaccharide of Haemophilus influenzae▿

The genes of the lic1 operon (lic1A to lic1D) are responsible for incorporation of phosphocholine (PCho) into the lipopolysaccharide (LPS) of Haemophilus influenzae. PCho plays a multifaceted role in the commensal and pathogenic lifestyles of a range of mucosal pathogens, including H. influenzae. Structural studies of the LPS of nontypeable H. influenzae (NTHI) have revealed that PCho can be linked to a hexose on any one of the oligosaccharide chain extensions from the conserved inner core triheptosyl backbone. In a collection of NTHI strains we found several strains in which there were two distinct but variant lic1D DNA sequences, genes predicted to encode the transferase responsible for directing the addition of PCho to LPS. The same isolates were also found to express concomitantly two PCho residues at distinct positions in their LPS. In one such NTHI isolate, isolate 1158, structural analysis of LPS from lic1 mutants confirmed that each of the two copies of lic1D directs the addition of PCho to a distinct location on the LPS. One position for PCho addition is a novel heptose, which is part of the oligosaccharide extension from the proximal heptose of the LPS inner core. Modification of the LPS by addition of two PCho residues resulted in increased binding of C-reactive protein and had consequential effects on the resistance of the organism to the killing effects of normal human serum compared to the effects of glycoforms containing one or no PCho. When bound, C-reactive protein leads to complement-mediated killing, indicating the potential biological significance of multiple PCho residues