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1.
Derek W. Hood rew D. Cox Michel Gilbert Katherine Makepeace Shannon Walsh Mary E. Deadman Alison Cody Adele Martin Martin Månsson Elke K. H. Schweda Jean-Robert Brisson James C. Richards E. Richard Moxon Warren W. Wakarchuk 《Molecular microbiology》2001,39(2):341-351
We have identified a gene for the addition of N- acetylneuraminic acid (Neu5Ac) in an α-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenza e. The gene is one that was identified previously as a phase-variable gene known as lic3A . Extracts of H. influenzae , as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N- acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae , Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118: lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-α-(2–3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118: lgtC lic3A , indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A , encoding an α-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene. 相似文献
2.
Schweda EK Landerholm MK Li J Richard Moxon E Richards JC 《Carbohydrate research》2003,338(23):2731-2744
Non-typeable Haemophilus influenzae (NTHi) is a significant cause of otitis media in children. We have employed single and multiple step electrospray ionization mass spectrometry (ESIMS) and NMR spectroscopy to profile and elucidate lipopolysaccharide (LPS) structural types expressed by NTHi strain 162, a strain obtained from an epidemiological study in Finland. ESIMS on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples of LPS provided information on the composition and relative abundance of glycoforms differing in the number of hexoses linked to the conserved inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A of H. influenzae LPS. The strain examined was found to elaborate Hex2 to Hex5 LPS glycoform populations having structures identical to those observed for H. influenzae strain Rd [Risberg, A.; Masoud, H.; Martin, A.; Richards, J.C.; Moxon, E.R.; Schweda, E.K.H. Eur. J. Biochem. 1999, 261, 171-180], the strain for which the complete genome has been sequenced. In addition, sialyllactose-containing glycoforms previously identified in strain Rd as well as several NTHi strains, were identified as minor components. Multiple step tandem ESIMS (MS(n)) on dephosphorylated and permethylated OS provided information on the arrangement of glycoses within the major population of glycoforms and on the existence of additional isomeric glycoforms. Minor Hex1 and Hex6 glycoforms were detected and characterized where the Hex6 glycoform was comprised of a dihexosamine-containing pentasaccharide chain attached at the proximal heptose residue of the inner-core unit. LPS structural motifs present in the NTHi strain 162 are expressed by a genetically diverse set of disease causing isolates, providing the basis for a vaccine strategy against NTHi otitis media. 相似文献
3.
Severi E Randle G Kivlin P Whitfield K Young R Moxon R Kelly D Hood D Thomas GH 《Molecular microbiology》2005,58(4):1173-1185
Sialylation of the lipopolysaccharide (LPS) is an important mechanism used by the human pathogen Haemophilus influenzae to evade the innate immune response of the host. We have demonstrated that N-acetylneuraminic acid (Neu5Ac or sialic acid) uptake in H. influenzae is essential for the subsequent modification of the LPS and that this uptake is mediated through a single transport system which is a member of the tripartite ATP-independent periplasmic (TRAP) transporter family. Disruption of either the siaP (HI0146) or siaQM (HI0147) genes, that encode the two subunits of this transporter, results in a complete loss of uptake of [14C]-Neu5Ac. Mutant strains lack sialylated glycoforms in their LPS and are more sensitive to killing by human serum than the parent strain. The SiaP protein has been purified and demonstrated to bind a stoichiometric amount of Neu5Ac by electrospray mass spectrometry. This binding was of high affinity with a Kd of approximately 0.1 microM as determined by protein fluorescence. The inactivation of the SiaPQM TRAP transporter also results in decreased growth of H. influenzae in a chemically defined medium containing Neu5Ac, supporting an additional nutritional role of sialic acid in H. influenzae physiology. 相似文献
4.
Fox KL Cox AD Gilbert M Wakarchuk WW Li J Makepeace K Richards JC Moxon ER Hood DW 《The Journal of biological chemistry》2006,281(52):40024-40032
The lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterized biochemically to date in H. influenzae is Lic3A, an alpha-2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., M?nsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both alpha-2,3- and alpha-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance. 相似文献
5.
Malin K Landerholm Jianjun Li James C Richards Derek W Hood E Richard Moxon Elke K H Schweda 《European journal of biochemistry》2004,271(5):941-953
Nontypeable Haemophilus influenzae (NTHi) is a common commensal of the human upper respiratory tract and is associated with otitis media in children. The structures of the oligosaccharide portions of NTHi lipopolysaccharide (LPS) from several otitis media isolates are now well characterized but it is not known whether there are structural differences in LPS from colonizing, nondisease associated strains. Structural analysis of LPS from nondisease associated NTHi strains 11 and 16 has been achieved by the application of high-field NMR techniques, ESI-MS, ESI-MSn, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. This is the first study to report structural details on LPS from strains taken from the nasopharynx from healthy individuals. Both strains express identical structures and contain the common element of H. influenzae LPS, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-lipid A, in which each heptose is elongated by a single hexose residue with no further oligosaccharide extensions. In the major Hex3 glycoform, the terminal Hepp residue (HepIII) is substituted at the O-2 position by a beta-D-Galp residue and the central Hepp residue (HepII) is substituted at O-3 by a alpha-D-Glcp residue. Notably, the strains express two phosphocholine (PCho) substituents, one at the O-6 position of alpha-D-Glcp and the other at the O-6 position of beta-D-Galp. Major acetylation sites were identified at O-4 of Gal and O-3 of HepIII. Additionally, both strains express glycine, and strain 11 also expresses detectable amounts of N-acetylneuraminic acid. 相似文献
6.
Hood DW Cox AD Gilbert M Makepeace K Walsh S Deadman ME Cody A Martin A Månsson M Schweda EK Brisson JR Richards JC Moxon ER Wakarchuk WW 《Molecular microbiology》2001,39(2):341-350
We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene. 相似文献
7.
D W Hood A D Cox W W Wakarchuk M Schur E K Schweda S L Walsh M E Deadman A Martin E R Moxon J C Richards 《Glycobiology》2001,11(11):957-967
A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)-]-L-alpha-D-Hepp-(1-->5)-alpha-Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D (1)H-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes involved in the addition of each glycose residue. 相似文献
8.
M M?nsson S H Bauer D W Hood J C Richards E R Moxon E K Schweda 《European journal of biochemistry》2001,268(7):2148-2159
Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486. 相似文献
9.
Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. Structural elucidation of the LPS from H. influenzae type b strain RM7004 was achieved by using electrospray ionization mass spectrometry (ESI-MS) and high-field NMR techniques on delipidated LPS and core oligosaccharide samples of LPS. It was found that the organism elaborates a series of related LPS glycoforms having a common inner-core structure, but differing in the number and position of attached hexose residues. LPS glycoforms containing between four and nine hexose residues were structurally characterized. The inner-core element was determined to be L-alpha-D-Hepp-(1-->2)-[PEA-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[P-->4]-alpha-KDOp-(2-->, a structural feature which has been identified in every H. influenzae strain investigated to date. Two major groups of isomeric glycoforms were characterized in which the terminal Hepp residue of the inner-core element was either substituted at the O-2 position with a beta-D-Galp residue or not. The structures of the major LPS glycoforms were found to have oligosaccharide chain extensions from O-3 of the middle Hepp residue. Glycoforms containing five and six hexose residues were most abundant and were shown to carry the tetrasaccharide unit alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->4)-alpha-D-Glcp at the O-3 position of the middle heptose. This tetrasaccharide displays the globoside trisaccharide (globotriose) as a terminal epitope, a structure that is found on many human cells (P(k) blood group antigen) and which is thought to be an important virulence determinant for H. influenzae. LPS glycoforms were characterized that had further chain extension from the beta-D-Glcp-(1--> residue of the proximal Hepp. In the fully extended LPS (Hex9/Hex8' glycoforms), both the proximal and middle heptose residues carried tetrasaccharide chains displaying terminal globotriose epitopes. In addition, the LPS was found to carry phosphorylcholine and O-acetyl groups. 相似文献
10.
Johnston JW Coussens NP Allen S Houtman JC Turner KH Zaleski A Ramaswamy S Gibson BW Apicella MA 《The Journal of biological chemistry》2008,283(2):855-865
Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4A resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium. 相似文献
11.
Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEA-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEA-->4]-alpha-Kdo-(2-->6)-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 alpha-d-Galp-(1-->4)-beta-d-Galp in both extensions, whereas lic2A directs only the expression of beta-d-Galp-(1-->4)-beta-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. 相似文献
12.
Yildirim HH Li J Richards JC Hood DW Moxon ER Schweda EK 《Carbohydrate research》2005,340(17):2598-2611
The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS. 相似文献
13.
Lipopolysaccharide (LPS) oligosaccharide epitopes are major virulence factors of Haemophilus influenzae. The structure of LPS glycoforms of H. influenzae type b strain Eagan containing a mutation in the gene lgtC is investigated. LgtC is involved in the biosynthesis of globoside trisaccharide [alpha-D-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-D-Glcp-(1-->], an LPS epitope implicated in the virulence of this organism. Glycose and methylation analyses provided information on the composition while electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS (LPS-OH) indicated the major glycoform to contain 4 hexoses attached to the common H. influenzae triheptosyl inner-core unit. The structure of the Hex4 glycoform in LPS-OH and core oligosaccharide samples was determined by NMR. It consists of an l-alpha-D-HepIIIp-(1-->2)-[PEtn-->6]-l-alpha-D-HepIIp-(1-->3)-l-alpha-D-HepIp-(1-->5)-[P-->4]-alpha-D-Kdop-(2--> to which a beta-D-Glcp-(1-->4)-alpha-D-Glcp disaccharide unit is extended from HepII at the C-3 position, while HepI and HepIII are substituted at the C-4 and C-2 positions with beta-D-Glcp and beta-D-Galp, respectively. This structure corresponds to that expressed as a subpopulation in the parent strain. 31P NMR studies permitted the identification of subpopulations of LPS containing Kdo substituted at the C-4 position with monophosphate or pyrophosphoethanolamine (PPEtn). HepIII was found to be substituted with either phosphate at the C-4 position or acetate at the C-3 position, but not both of them together in the same subpopulation. The subpopulations containing phosphate and acetate at HepIII and their location have not previously been reported. 相似文献
14.
Bauer SH Månsson M Hood DW Richards JC Moxon ER Schweda EK 《Carbohydrate research》2001,335(4):251-260
In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes. 相似文献
15.
The structure for the carbohydrate moiety of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 129Pt was elucidated. The structure of the core oligosaccharide and O-deacylated LOS was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the major fully extended carbohydrate glycoform of the LOS was determined on the basis of the combined data from these experiments. [Carbohydrate structure: see text]. In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and PEtn is phosphoethanolamine. Minor amounts of glycoforms containing nonstoichiometric substituents glycine and phosphate at the distal heptose residue were also identified. 相似文献
16.
E K Schweda J R Brisson G Alvelius A Martin J N Weiser D W Hood E R Moxon J C Richards 《European journal of biochemistry》2000,267(12):3902-3913
Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and 1H NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1--> 3)-L-alpha-D-He pp-(1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI. 相似文献
17.
The structure of the lipopolysaccharide of Haemophilus influenzae mutant strain, RM.118-26, was investigated. Electrospray ionization-mass spectrometry on intact lipopolysaccharide, O-deacylated lipopolysaccharide and core oligosaccharides obtained from lipopolysaccharide after mild acid hydrolysis provided information on the composition and relative abundance of the glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. The structure of the major glycoform containing phosphocholine is identical to the Hex2 glycoform described for H. influenzae RM.118-28 [Risberg, A., Schweda, E.K.H. & Jansson, P.-E. (1997) Eur. J. Biochem. 243, 701-707]. A second major glycoform, containing three hexose residues (Hex3), in which a lactose unit, beta-D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-( 1-->4)-]- L-alpha-D-Hepp-(1-->5)-alpha-Kdo, carries no phosphocholine. Instead this lipopolysaccharide glycoform is partly (40%) substituted by an O-acetyl group linked to the 6-position of the glucose residue in the lactose unit and has the following structure: 相似文献
18.
A Risberg H Masoud A Martin J C Richards E R Moxon E K Schweda 《European journal of biochemistry》1999,261(1):171-180
Structural elucidation of the lipopolysaccharide (LPS) of Haemophilus influenzae, strain Rd, a capsule-deficient type d strain, has been achieved by using high-field NMR techniques and electrospray ionization-mass spectrometry (ESI-MS) on delipidated LPS and core oligosaccharide samples. It was found that this organism expresses heterogeneous populations of LPS of which the oligosaccharide (OS) epitopes are subject to phase variation. ESI-MS of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues linked to a conserved inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp- (1-->4)-]- L-alpha-D-Hepp-(1-->5)-alpha-Kdo, and the degree of phosphorylation. The structures of the major LPS glycoforms containing three (two Glc and one Gal), four (two Glc and two Gal) and five (two Glc, two Gal and one GalNAc) hexoses were substituted by both phosphocholine (PCho) and phosphoethanolamine (PEtn) and were determined in detail. In the major glycoform, Hex3, a lactose unit, beta-D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner-core element. The Hex4 glycoform contains the PK epitope, alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp while in the Hex5 glycoform, this OS is elongated by the addition of a terminal beta-D-GalpNAc residue, giving the P antigen, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-D-Glc p. The fully extended LPS glycoform (Hex5) has the following structure. [see text] The structural data provide the first definitive evidence demonstrating the expression of a globotetraose OS epitope, the P antigen, in LPS of H. influenzae. It is noteworthy that the molecular environment in which PCho units are found differs from that observed in an Rd- derived mutant strain (RM.118-28) [Risberg, A., Schweda, E. K. H. & Jansson, P-E. (1997) Eur. J. Biochem. 243, 701-707]. 相似文献
19.
Lundström SL Twelkmeyer B Sagemark MK Li J Richards JC Hood DW Moxon ER Schweda EK 《The FEBS journal》2007,274(18):4886-4903
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 [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-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-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-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. 相似文献
20.
Capillary electrophoresis (CE) is a high-resolution separation technique that has been widely used for trace analysis in biological samples. On-line capillary electrophoresis-electrospray mass spectrometry (CE-MS) was developed for the analysis of lipopolysaccharide (LPS) glycoforms from the gram-negative bacteria, Haemophilus influenzae. In this paper, we report on the application of CE-MS to characterize structural differences in O-deacylated LPS samples from H. influenzae strains Rd 11.7 and 375.1. The resolution capability of on-line CE-MS was first demonstrated by analysis of a complex LPS mixture from H. influenzae strain Rd 11.7. This strain contains a mixture of isomeric glycoforms differing in the number and positions of hexose moieties. Sialic acid containing glycoforms were also determined. Structural features of LPS from a lic1 mutant of H. influenzae strain 375 (375.1) were studied using on-line CE-MS/MS. With the separation provided by CE, two isomeric glycoforms differing in the location of phosphoethanolamine substituents were characterized by tandem mass spectrometry. 相似文献