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-MSⁿ 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 β-d-Galp-(1→4)-β-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 β-d-Glcp which, in turn, can be further extended. Contrary to previous findings, LPS of the pgm mutant strain contained minor glycoforms having β-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.     

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)-β-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses 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-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and lactose [β-d-Galp-(1→4)-β-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)-β-d-Gal-(1→4)-β-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.     

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-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-GlcIp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdop) to which addition of β-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 β-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 β-d-Galp-(1→4-β-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 ESIMSⁿ on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β-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.     

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 β-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 β-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.     

Structural profiling of lipopolysaccharide glycoforms expressed by non-typeable Haemophilus influenzae: phenotypic similarities between NTHi strain 162 and the genome strain Rd

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.     

Structural elucidation of the major Hex4 lipopolysaccharide glycoform from the lgtC mutant of Haemophilus influenzae strain Eagan

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.     

Novel O-antigen of Hafnia alvei PCM 1195 lipopolysaccharide with a teichoic acid-like structure

The lipopolysaccharide (LPS) of Hafnia alvei strain PCM 1195 was obtained by the hot phenol/water method. The O-specific polysaccharide was released by mild acidic hydrolysis and isolated by gel filtration. The structure of the O-specific polysaccharide was investigated by ¹H, ¹³C, and ³¹P NMR spectroscopy, MALDI-TOF MS, and GC-MS, accompanied by monosaccharide and methylation analysis. It was concluded that the O-specific polysaccharide is composed of a hexasaccharide repeating units interlinked with a phosphate group: {→4-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-[α-d-Glcp-(1→4)]-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→4)-α-d-Glcp-(1→P}_n.     

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.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176

The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core 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 having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [M?nsson, M.; Bauer, S. H. J.; Hood, D. W.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H., Eur. J. Biochem. 2001, 268, 2148--2159]. The following LPS structures were identified as the major glycoforms, the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep):     

An acetylated galactomannoglucan from the stems of Dendrobium nobile Lindl

A water-soluble polysaccharide DNP-W2 composed of glucose, mannose, and galactose in the molar ratio of 6.1:2.9:2.0 had been isolated from the stems of Dendrobium nobile. Its molecular weight was 1.8 × 10⁴ Da determined by HPGPC. Structural features of DNP-W2 were investigated by a combination of chemical and instrumental analysis, including FTIR, GC, GC-MS, periodate oxidation-Smith degradation, methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The results showed that DNP-W2 is a 2-O-acetylgalactomannoglucan and has a backbone consisting of (1→4)-linked β-d-Glcp, (1→6)-linked β-d-Glcp, and (1→4)-linked β-d-Manp, with branches at O-6 of (1→4)-linked β-d-Glcp and β-d-Manp. The branches are composed of α-d-Galp. The acetyl groups are substituted at O-2 of (1→4)-linked Manp. Preliminary tests in vitro reveals that DNP-W2 can stimulate ConA- and LPS-induced T and B lymphocyte proliferation.     

Structural investigation of lipopolysaccharides from nontypeable Haemophilus influenzae: investigation of inner-core phosphoethanolamine addition in NTHi strain 981

LPS of NTHi 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-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-alpha-Kdop) in which addition of PEtn to the central heptose (HepII) in strain Rd is controlled by the gene lpt6. It was recently shown that NTHi strain 981 contains an additional PEtn linked to O-3 of the terminal heptose of the inner-core moiety (HepIII). In order to establish whether lpt6 is also involved in adding PEtn to HepIII, lpt6 in strain 981 was inactivated. The structure of the LPS of the resulting mutant strain 98llpt6 was investigated by MS and NMR techniques by which it was confirmed that the lpt6 gene product is responsible for addition of PEtn to O-6 of HepII in strain 981. However, it is not responsible for adding PEtn to O-3 of HepIII since the 981lpt6 mutant still had full substitution with PEtn at HepIII.     

Structure of the high-molecular weight exopolysaccharide isolated from Lactobacillus pentosus LPS26

The strain Lactobacillus pentosus LPS26 produces a capsular polymer composed of a high- (2.0 × 10⁶ Da) (EPS A) and a low-molecular mass (2.4 × 10⁴ Da) (EPS B) polysaccharide when grown on semi-defined medium containing glucose as the carbon source. The structure of EPS A and its deacetylated form has been determined by monosaccharide and methylation analysis as well as by 1D/2D NMR studies (¹H and ¹³C). We conclude that EPS A is a charged heteropolymer, with a composition of d-glucose, d-glucuronic acid and l-rhamnose in a molar ratio 1:2:2. The repeating unit is a pentasaccharide with two O-acetyl groups at O-4 of the 3-substituted α-d-glucuronic acid and at O-2 of the 3-substituted β-l-rhamnose, respectively.→4)-α-d-Glcp-(1→3)-α-d-GlcpA4Ac-(1→3)-α-l-Rhap-(1→4)-α-d-GlcpA-(1→3)-β-l-Rhap2Ac-(1→This unbranched structure is not common in EPSs produced by Lactobacilli. Moreover, the presence of acetyl groups in the structure is an unusual feature which has only been reported in L. sake 0-1 [Robijn et al. Carbohydr. Res., 1995, 276, 117-136].     

Glycan flexibility: insights into nanosecond dynamics from a microsecond molecular dynamics simulation explaining an unusual nuclear Overhauser effect

An atomistic all-atom molecular dynamics simulation of the trisaccharide β-d-ManpNAc-(1→4)[α-d-Glcp-(1→3)]-α-l-Rhap-OMe with explicit solvent molecules has been carried out. The trisaccharide represents a model for the branching region of the O-chain polysaccharide of a strain from Aeromonas salmonicida. The extensive MD simulations having a 1-μs duration revealed a conformational dynamics process on the nanosecond time scale, that is, a ‘time window’ not extensively investigated for carbohydrates to date. The results obtained from the MD simulation underscore the predictive power of molecular simulations in studies of biomolecular systems and also explain an unusual nuclear Overhauser effect originating from conformational exchange.     

Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd.

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].     

Structure of the O-polysaccharide of the lipopolysaccharide of Pragia fontium 97U116

The O-polysaccharide of Pragia fontium 97U116 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the pentasaccharide-repeating unit was established: →2)-α-d-Galf-(1→3)-α-l-Rhap2Ac^I-(1→4)-α-d-GlcpNAc^I-(1→2)-α-l-Rhap^II-(1→3)-β-d-GlcpNAc^II-(1→     

Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142

This paper describes the structure of neutral exopolysaccharide (EPS) produced by Lactobacillus johnsonii 142, strain of the lactic acid bacteria isolated from the intestine of mice with experimentally induced inflammatory bowel disease (IBD). Sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ¹H,¹H COSY, TOCSY, NOESY, and ¹H,¹³C HSQC experiments revealed that the repeating unit of the EPS is a pentasaccharide:→3)-α-d-Galp-(1→3)-β-d-Glcp-(1→5)-β-d-Galf-(1→3)-α-d-Galp-(1→3)-α-d-Galp-(1→The rabbit antiserum raised against whole cells of L. johnsonii 142 reacted with homologous EPS, and cross-reacted with exopolysaccharide from Lactobacillus animalis/murinus 148 isolated also from mice with IBD, but not reacted with EPS of L. johnsonii 151 from healthy mice.     

Structure of a polysaccharide from the lipopolysaccharides of Vibrio vulnificus strains CECT 5198 and S3-I2-36, which is remarkably similar to the O-polysaccharide of Pseudoalteromonas rubra ATCC 29570

High-molecular-mass polysaccharides were released by mild acid degradation of the lipopolysaccharides of two wild-type Vibrio vulnificus strain, a flagellated motile strain CECT 5198 and a non-flagellated non-motile strain S3-I2-36. Studies by sugar analysis and partial acid hydrolysis along with ¹H and ¹³C NMR spectroscopies showed that the polysaccharides from both strains have the same trisaccharide repeating unit of the following structure:→4)-β-d-GlcpNAc3NAcylAN-(1→4)-α-l-GalpNAmA-(1→3)-α-d-QuipNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose, GalNAmA for 2-acetimidoylamino-2-deoxygalacturonic acid, GlcNAc3NAcylAN for 2-acetamido-3-acylamino-2,3-dideoxyglucuronamide and acyl for 4-d-malyl (∼30%) or 2-O-acetyl-4-d-malyl (∼70%). The structure of the polysaccharide studied resembles much that of a marine bacterium Pseudoalteromonas rubra ATCC 29570 reinvestigated in this work. The latter differs in (i) the absolute configuration of malic acid (l vs d), (ii) 3-O-acetylation of GalNAmA and (iii) replacement of QuiNAc with its 4-keto biosynthetic precursor.     

Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation

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.     

Structure of the O-polysaccharide of Salmonella enterica O41

An O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Salmonella enterica O41, and the following structure of the O-unit was determined by chemical analyses along with 1D and 2D ¹H and ¹³C NMR spectroscopy:→2)-β-d-Manp-(1→4)-α-d-Glcp-(1→3)-α-l-QuipNAc-(1→3)-α-d-GlcpNAc-(1→where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose. The structure established is in agreement with the O-antigen gene cluster of S. enterica O41 and tentative assignment of the gene functions reported earlier.