Structural analysis of the lipooligosaccharide from the commensal Haemophilus somnus genome strain 129Pt

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.     

Structural analysis of the oligosaccharide of Histophilus somni (Haemophilus somnus) strain 2336 and identification of several lipooligosaccharide biosynthesis gene homologues

The structure of the core oligosaccharide from a pneumonic Histophilus somni (Haemophilus somnus) strain 2336 was elucidated. The lipooligosaccharide (LOS) was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core oligosaccharide was determined on the basis of the combined data from these experiments: [formula-see text]. The structural elucidation was intriguing as it suggested several differences in the LOS structures between strain 2336 and the related strain 738. Strain 738 originated following passaging of strain 2336 through a calf. The differences between the two structures are a different linkage between Gal II and GlcNAc (1-->4 here; 1-->3 in 738), the absence of phosphocholine (PCho) from 2336 and the presence of two phosphoethanolamine (PEtn) residues and Gal III (at the 2-position) of Hep II in 2336. Although pulse-field gel electrophoresis data following digest with only one restriction enzyme showed identical profiles suggesting that strains 738 and 2336 are the same strain, the structural data does suggest that, if strain 738 is indeed a phase variant of strain 2336, considerable variation occurred on calf passaging and could therefore be an intriguing example of how broadly this bacterium can adapt itself in the host.     

Structural analysis of the lipooligosaccharide-derived oligosaccharide of Histophilus somni (Haemophilus somnus) strain 8025

Previous structural studies in our laboratory on lipooligosaccharide (LOS) inner core oligosaccharide (OS) had identified structures from several strains of Histophilus (Haemophilus) somni (738, 2336, 1P, 129Pt). Recently a type strain 8025 was proposed for this species and we therefore sought to determine the core OS structure of this H. somni strain. Core OS was isolated by standard methods from Westphal purified LOS. Structural information was established by a combination of monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core OS was determined on the basis of the combined data from these experiments: [carbohydrates: see text]. The structure determined contains aspects of other Histophilus somni core OS structures, such as the beta-Gal attached at the 2-position of Hep II (2336), PEtn only at the 6-position of Hep II (738, 129Pt) and a lactose extension from Hep I (1P). Since genetic manipulation has been achieved with this strain, the identification of the core OS structure will enable experiments designed to identify the role of glycosyltransferases involved in LOS biosynthesis.     

Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae.

Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.     

The structure of the core region of the lipopolysaccharide from Shewanella algae BrY, containing 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid

The structure of the carbohydrate backbone of the lipid A-core region of the LPS from Shewanella algae strain BrY was analysed. The LPS was N,O-deacylated to give three products, which were isolated and studied by chemical methods, NMR and mass spectrometry: [Carbohydrate structures: see text]. All monosaccharides except L-rhamnose had the D-configuration. This LPS presents a second example (after S. oneidensis) of the structure with a novel linking unit between the core and lipid A moieties, 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid (8-amino-Kdo).     

Structural studies on the exopolysaccharide from Erwinia persicina

The Gram-negative bacterial strain HKI 0380 was isolated from biofilms located on palaeolithic rock paintings in the Cave of Bats in Zuheros, southern Spain. It was identified as the phytopathogenic Erwinia persicina and attracted attention due to the production of considerable quantities of slime. The acidic exopolysaccharide produced by the E. persicina was studied after O-deacylation by sugar and methylation analyses, along with (1)H and (13)C NMR spectroscopy. The following structure of the branched pentasaccharide repeating unit of the O-deacylated exopolysaccharide was established: [carbohydrate structure: see text].     

Cloning and characterization of the lipooligosaccharide galactosyltransferase II gene of Haemophilus ducreyi

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.     

Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.     

Sequence-based predictions of lipooligosaccharide diversity in the Neisseriaceae and their implication in pathogenicity

Endotoxin [Lipopolysaccharide (LPS)/Lipooligosaccharide (LOS)] is an important virulence determinant in gram negative bacteria. While the genetic basis of endotoxin production and its role in disease in the pathogenic Neisseria has been extensively studied, little research has focused on the genetic basis of LOS biosynthesis in commensal Neisseria. We determined the genomic sequences of a variety of commensal Neisseria strains, and compared these sequences, along with other genomic sequences available from various sequencing centers from commensal and pathogenic strains, to identify genes involved in LOS biosynthesis. This allowed us to make structural predictions as to differences in LOS seen between commensal and pathogenic strains. We determined that all neisserial strains possess a conserved set of genes needed to make a common 3-Deoxy-D-manno-octulosonic acid -heptose core structure. However, significant genomic differences in glycosyl transferase genes support the published literature indicating compositional differences in the terminal oligosaccharides. This was most pronounced in commensal strains that were distally related to the gonococcus and meningococcus. These strains possessed a homolog of heptosyltransferase III, suggesting that they differ from the pathogenic strains by the presence a third heptose. Furthermore, most commensal strains possess homologs of genes needed to synthesize lipopolysaccharide (LPS). N. cinerea, a commensal species that is highly related to the gonococcus has lost the ability to make sialyltransferase. Overall genomic comparisons of various neisserial strains indicate that significant recombination/genetic acquisition/loss has occurred within the genus, and this muddles proper speciation.     

Investigation of the structural heterogeneity of lipooligosaccharides from pathogenic Haemophilus and Neisseria species and of R-type lipopolysaccharides from Salmonella typhimurium by electrospray mass spectrometry.

Heterogeneity in the lipooligosaccharides (LOS) of pathogenic Haemophilus and Neisseria species is evident from the multiplicity of components observed with electrophoretic analyses. Knowledge of the precise structures that make up these diverse LOS molecules is clearly the key to reaching an understanding of pathogenic processes such as phase variation and molecular mimicry. Except for a few cases, little is known about the specific structural features of LOS that underlie phase variation and molecular mimicry, partly because of the inherent difficulties in the structural elucidation of these complex glycolipids. In the lipopolysaccharides (LPS) from Salmonella typhimurium and Escherichia coli, rough, or R-type, mutants have been isolated that have provided insight into the biosynthetic pathways and associated genetics that control LPS expression. Nonetheless, recent work has shown that these R-type LPS are more complex than originally thought, and significant heterogeneity is still observed, primarily in their phosphorylation states. In order to investigate the structures of LPS and LOS in a more rapid fashion, we have determined the precise molecular weights of LOS (and LPS) preparations from various Haemophilus, Neisseria, and Salmonella species by electrospray ionization-mass spectrometry. The LOS (or LPS) were first O-deacylated under mild hydrazine conditions to remove O-linked esters primarily from the lipid A portion. Under negative-ion conditions, the O-deacylated LOS yield abundant multiply deprotonated molecular ions, (M-nH)n-, where n refers to the number of protons removed and therefore determines the absolute charge state, n = z. Mass spectra from different LOS and LPS preparations have provided detailed information concerning the structural basis for LOS (and LPS) heterogeneity and corresponding saccharide compositions. The identification of sialic acid in the LOS of Haemophilus and Neisseria species and the variable phosphorylation of the core of S. typhimurium LPS have afforded insights into the biosynthetic pathways used by these organisms. Information of this type is important for understanding the underlying genetic and environmental factors controlling LOS and LPS expression.     

Characterization of the N-acetyl-5-neuraminic acid-binding site of the extracytoplasmic solute receptor (SiaP) of nontypeable Haemophilus influenzae strain 2019

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.     

Structural and serological characterisation of the O-antigenic polysaccharide of the lipopolysaccharide from Acinetobacter baumannii strain 24

Extraction of dry bacteria of Acinetobacter baumannii strain 24 by phenol-water yielded a lipopolysaccharide (LPS) that was studied by serological methods and fatty acid analysis. After immunisation of BALB/c mice with this strain, monoclonal antibody S48-3-13 (IgG(3) isotype) was obtained, which reacted with the LPS in western blot and characterized it as S-form LPS. Degradation of the LPS in aqueous 1% acetic acid followed by GPC gave the O-antigenic polysaccharide, whose structure was determined by compositional analyses and NMR spectroscopy of the polysaccharide and O-deacylated polysaccharide as [carbohydrate structure: see text] where QuiN4N is 2,4-diamino-2,4,6-trideoxyglucose and GalNAcA 2-acetamido-2-deoxygalacturonic acid. The amino group at C-4 of the QuipN4N residues is acetylated in about 2/3 of LPS molecules and (S)-3-hydroxybutyrylated in the rest.     

Structural analysis of the lipopolysaccharide from Neisseria meningitidis strain BZ157 galE: localisation of two phosphoethanolamine residues in the inner core oligosaccharide

The structure of the phase-variable lipopolysaccharide (LPS) from the group B Neisseria meningitidis strain BZ157 galE was elucidated. The structural basis for the LPS's variation in reactivity with a monoclonal antibody (MAb) B5 that has specificity for the presence of phosphoethanolamine (PEtn) at the 3-position of the distal heptose residue (HepII) was established. The structure of the O-deacylated LPS was deduced by a combination of monosaccharide analyses, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These analyses revealed the presence of a novel inner core oligosaccharide (OS) structure in the MAb B5 reactive (B5+) LPS that contained two PEtn residues simultaneously substituting the 3- and 6-positions of the HepII residue. The determination of this structure has identified a further degree of variability within the inner core OS of meningococcal LPS that could contribute to the interaction of meningococcal strains with their host.     

Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd

Haemophilus somnus can be either a commensal of bovine mucosal surfaces or an opportunistic pathogen. Pathogenic strains of H. somnus are a significant cause of systemic disease in cattle. We report the genome sequence of H. somnus 129Pt, a nonpathogenic commensal preputial isolate, and the results of a genome-wide comparative analysis of H. somnus 129Pt, Haemophilus influenzae Rd, and Haemophilus ducreyi 35000HP. We found unique genes in H. somnus 129Pt involved in lipooligosaccharide biosynthesis, carbohydrate uptake and metabolism, cation transport, amino acid metabolism, ubiquinone and menaquinone biosynthesis, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport. There were also many genes in common among the three organisms. Our comparative analyses of H. somnus 129Pt, H. influenzae Rd, and H. ducreyi 35000HP revealed similarities and differences in the numbers and compositions of genes involved in metabolism, host colonization, and persistence. These results lay a foundation for research on the host specificities and niche preferences of these organisms. Future comparisons between H. somnus 129Pt and virulent strains will aid in the development of protective strategies and vaccines to protect cattle against H. somnus disease.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

Phase variation of a beta-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni

Ganglioside mimicry by Campylobacter jejuni lipo-oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain-Barré and Miller-Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1-like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside-mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal beta-1,3-linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a beta-1,3 galactosyltransferase responsible for converting GM2-like LOS structures to GM1-like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside-mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1-like LOS structure as well as the ganglioside GM2-like LOS structure proposed in the literature.     

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.     

The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.     

Structural determination of the O-antigenic polysaccharide from the Shiga toxin-producing Escherichia coli O171

The structure of the O-antigenic part of the lipopolysaccharide (LPS) obtained from the verotoxin-producing Escherichia coli O171 has been determined. (1)H and (13)C NMR spectroscopy techniques in combination with component analysis were used to elucidate the O-antigen structure of O-deacylated LPS. Subsequent NMR analysis of the native LPS revealed acetylation at O-7/O-9 of the sialic acid residue. The sequence of sugars was determined by inter-residue correlations in (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation spectra. The O-antigen is composed of pentasaccharide repeating units with one equivalent of O-acetyl groups distributed over two positions: -->4)-alpha-Neu5Ac7,9Ac-(2-->6)-beta-D-Galp-(1-->6)-beta-DGlcp-->(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1--> Based on biosynthetic considerations, this should also be the biological repeating unit.     

The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549T

Novel lipooligosaccharide components were isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Pseudoalteromonas issachenkonii type strain KMM 3549T. The complete structure was achieved by chemical analysis, 2D NMR spectroscopy and MALDI mass spectrometry as the following: [carbohydrate formula see text] All sugars are d-pyranoses. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked. In addition, by MALDI mass spectrometry of the intact LOS, the lipid A moiety was also identified as a mixture of penta-, tetra- and triacylated species.