The molecular mechanism of phase variation of H. influenzae lipopolysaccharide

Multiple carbohydrate structures on the outer-membrane lipopolysaccharide (LPS) of the gram-negative pathogen H. influenzae undergo high frequency, reversible loss, indicative of phase variation. Characterization of a genetic locus, lic-1, responsible for expression of two LPS epitopes displaying phase variation, showed it to comprise four genes. The first gene mediates phase variation. At its 5' end, within the open reading frame, are a variable number of tandem repeats of the tetramer CAAT. By shifting upstream initiation codons in or out of frame, these 4 bp units create a translational switch. The phenotype of organisms corresponds to the number of 4 bp units. Phase variation between three levels of expression ( +, +, and -) of lic-1-derived epitopes is caused by differences in the three phases of translation of the 5' terminus of this gene. Phase variation also allows for selection of organisms displaying certain LPS epitopes in vivo.     

Intra-strain variation in expression of lipopolysaccharide by Helicobacter pylori

Intra-strain variation in the expression of lipopolysaccharide (LPS) by two clinical isolates of Helicobacter pylori was examined. Lipopolysaccharide was prepared from successive cultures of individual colonies from each strain, separated by SDS-PAGE, and detected by silver staining and by immunoblotting. The genetic 'relatedness' of the colonies was investigated using PCR-RFLP analysis of the urease and vacuolating cytotoxin genes. Although individual colonies of each of the two strains examined appeared to have the same genetic origins, variation in the expression of their long-chain LPS was observed. The same LPS profiles were maintained by individual colonies over four subcultures on solid media containing 10% (v/v) defibrinated horse blood.     

The role of a repetitive DNA motif (5'-CAAT-3') in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope αGal(1–4)βGal

Haemophilus influenzae lipopolysaccharide (LPS) contains structures, defined by monoclonal antibodies, which undergo phase variation. This investigation reports the nucleotide sequence of lic2A, which is required for the expression of at least three phase-variable LPS epitopes, one of which has the structure αGal(1–4)βGal. lic2A contains multiple tandem repeats of the tetramer 5′-CAAT-3′ Previous studies have correlated changes in the number of 5′-CAAT-3′ repeats with the phase-variable expression of the αGal(1–4)βGal epitope. To obtain direct evidence for this, the 5′-CAAT-3′ repeat region from lic2A was amplified directly from immunostained colonies and sequenced. This demonstrated that the variable expression of LPS epitopes, including αGal(1–4)βGal, is in part directly dependent upon the number of copies of 5′-CAAT-3′ within lic2A.     

Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae.

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.     

Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis

Lipopolysaccharide (LPS) is a major determinant of Neisseria meningitidis virulence. A key feature of meningococcal LPS is the phase-variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase-variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae , was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic-resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine—SDS—PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto- N -neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5'-end of the coding sequence. As the lacto- N -neotetraose structure in meningococcal LPS is subject to phase-variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an 'off' phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped-strand mispairing mechanism, operating in the first ORF, controls the phase-variable expression of lacto- N -neotetraose.     

Adaptation of Haemophilus influenzae to acquired and innate humoral immunity based on phase variation of lipopolysaccharide

Phase variation in colony morphology has been associated with the pathogenesis of infection caused by Haemophilus influenzae. This study shows that differences in colony opacity in non-typeable H. influenzae (NTHi) strain H233 involve phase changes in the lipopolysaccharide (LPS) and depend on the expression of licl and lic2, which contain translational switches based on intragenic tandem repeats of 5'-CAAT-3'. Genetic analysis showed that opaque organisms have an out-of-frame number of repeats in both licl, required for the expression of phosphorylcholine (ChoP), and lic2, a putative galactosyl transferase that adds the terminal galactose on Galalpha1-4Gal. Defined variants in these loci were used to examine the contribution of individual LPS structures to resistance to serum bactericidal activity mediated by antibody and C-reactive protein (CRP). The addition of ChoP by licl was the only factor in serum killing involving CRP and complement. The terminal galactose moiety, in contrast, conferred resistance to killing by naturally acquired antibody and complement present in human serum. As Galalpha1-4Gal is also found on human glycolipids, it appears that decoration of the cell surface with this host-like antigen blocks antibody-mediated serum bactericidal activity. Genetic analysis of NTHi within the human respiratory tract demonstrated that Galalpha1-4Gal may not be expressed during carriage but may be advantageous for the organism in inflammatory states such as pneumonia.     

Characterization of repetitive sequences controlling phase variation of Haemophilus influenzae lipopolysaccharide.

Phase variation of lipopolysaccharide epitopes of an Haemophilus influenzae serotype b strain (strain RM.7004) occurs through a mechanism which depends on multiple tandem repeats of the DNA sequence 5'-CAAT-3' situated within the chromosomal locus lic1. We report here that the same tetranucleotide repeats are also found in two other genomic loci (lic2 and lic3) of RM.7004. Similar to lic1, there are multiple tandem repeats of 5'-CAAT-3' present at the 5' ends of long open reading frames in lic2 and lic3. Variation in the number of repeats of CAAT, by shifting the upstream initiation codons in or out of phase with the remainder of the open reading frame, could switch on or off the translation of downstream genes. Similar to previously reported findings for lic1, site-directed mutations in the open reading frame downstream (3') from the repeats of CAAT in lic2 abolished phase variation and identified DNA sequences required for the expression of additional oligosaccharide epitopes. When we used an oligonucleotide comprising five repeats of CAAT or DNA sequences specific for lic1, lic2, and lic3 as probes, a survey of other encapsulated H. influenzae strains (serotypes a through f) and nontypable H. influenzae strains (including biotype aegyptius) showed that the chromosome of H. influenzae can have from two to five regions which contain multiple tandem repeats of CAAT in addition to other sequences which hybridize to lic1 and lic2.     

The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein-mediated killing

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C-reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP-mediated serum bactericidal activity regardless of the genetic background. Differences in CRP-mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.     

Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide

Phosphorylcholine (ChoP) is a common surface feature of many mucosal organisms, including Neisseria spp., in which it is present exclusively on pili of pathogenic Neisseria and on the lipopolysaccharide (LPS) of commensal Neisseria (Cn). Its presence in Cn has been confirmed by nuclear magnetic resonance. It appears that choline is the main source for the production of ChoP by Cn. We have sequenced a locus, containing four genes (licA-D) with 47-73% identity to the lic1 locus of Haemophilus influenzae (Hi) and 21-40% identity to lic genes in Streptococcus pneumoniae, involved in the production and incorporation of ChoP. The arrangement of the Cn genes and the presence of CAAT repeats, responsible for phase variation of ChoP expression, resemble Hi and differ from S. pneumoniae. Cn DNA flanking the lic locus contains genes ilvE and NMA2149 with >85% identity to the pathogenic Neisseria genes. However, there are no lic genes in the corresponding location or elsewhere in pathogenic Neisseria. This suggests either the loss of the locus from pathogenic Neisseria or a horizontal transfer of genes to Cn, perhaps from H. influenzae spp. As in Hi, ChoP enhances adherence to and invasion of human epithelial cells via the receptor for platelet-activating factor. However, ChoP expression also increases susceptibility to serum killing mediated by complement and C-reactive protein. Taken together, these observations support the hypothesis that the ability of many organisms to switch off ChoP expression rapidly represents an important adaptation to different environments encountered during the colonization/infection process and that the ChoP moiety apparently synthesized by distinct means in pathogenic and commensal Neisseria represents an advantage in the colonization properties of these bacteria.     

Novel lipopolysaccharide biosynthetic genes containing tetranucleotide repeats in Haemophilus influenzae, identification of a gene for adding O-acetyl groups

Many of the genes for lipopolysaccharide (LPS) biosynthesis in Haemophilus influenzae are phase variable. The mechanism of this variable expression involves slippage of tetranucleotide repeats located within the reading frame of these genes. Based on this, we hypothesized that tetranucleotide repeat sequences might be used to identify as yet unrecognized LPS biosynthetic genes. Synthetic oligonucleotides (20 bases), representing all previously reported LPS-related tetranucleotide repeat sequences in H. influenzae, were used to probe a collection of 25 genetically and epidemiologically diverse strains of non-typeable H. influenzae. A novel gene identified through this strategy was a homologue of oafA, a putative O-antigen LPS acetylase of Salmonella typhimurium, that was present in all 25 non-typeable H. influenzae, 19 of which contained multiple copies of the tetranucleotide 5'-GCAA. Using lacZ fusions, we showed that these tetranucleotide repeats could mediate phase variation of this gene. Structural analysis of LPS showed that a major site of acetylation was the distal heptose (HepIII) of the LPS inner-core. An oafA deletion mutant showed absence of O-acetylation of HepIII. When compared with wild type, oafA mutants displayed increased susceptibility to complement-mediated killing by human serum, evidence that O-acetylation of LPS facilitates resistance to host immune clearance mechanisms. These results provide genetic and structural evidence that H. influenzae oafA is required for phase variable O-acetylation of LPS and functional evidence to support the role of O-acetylation of LPS in pathogenesis.     

Phosphorylcholine decoration of lipopolysaccharide differentiates commensal Neisseriae from pathogenic strains: identification of licA-type genes in commensal Neisseriae

Phosphorylcholine (ChoP) is a potential candidate for a plurispecific vaccine, because it is present on surface components of many mucosal organisms, including Haemophilus influenzae, Streptococcus pneumoniae and Pseudomonas aeruginosa. In addition, ChoP has been detected on pili of Neisseria meningitidis and Neisseria gonorrhoeae. In this study, we demonstrate the presence of the phosphorylcholine epitope on the lipopolysaccharides (LPSs) of several species of commensal Neisseriae (Cn), a property that differentiates commensal from the pathogenic strains of Neisseriae. In an extended survey of 78 strains, we confirmed the exclusive expression of the ChoP epitope on pili of pathogenic Neisseriae. Despite the presence of pili on Cn, which are homologous to Class II pili of N. meningitidis, they did not react with anti-ChoP antibody. This observation was further supported by the fact that 14C-labelled choline was incorporated only in the LPSs of Cn. Analysis of the LPS of N. lactamica strain NL4 revealed two distinct and interconvertible molecular species of LPS with high and low levels of reactivity with anti-ChoP antibody. In addition, on/off phase variation gave rise to frequent modulation in the levels of antibody reactivity. A concurrent modulation was also observed in the binding of C-reactive protein, CRP, a ChoP-binding reactant that is implicated in bacterial clearance. Genetic analysis showed the presence of a gene in several Cn spp. with significant sequence identity to H. influenzae licA. This gene encodes choline kinase and is also involved in phase variation of the LPS-associated ChoP in H. influenzae. In contrast, licA-like genes were not identified in the pathogenic Neisseria strains tested. They are absent from N. meningitidis strain Z2491 genome database. These data suggest that the genetic basis for ChoP incorporation in Cn LPS resembles that in H. influenzae spp. and may be distinct from that generating the ChoP epitope on pili of pathogenic Neisseriae. Further, the modulation of ChoP expression on Cn LPS, and corresponding modulation of CRP binding, has the potential to confer the property of immune avoidance and thus of persistence on mucosa.     

The role of licA phase variation in the pathogenesis of invasive disease by Haemophilus influenzae type b

LicA encodes the enzyme phosphorylcholine kinase which catalyses the incorporation of phosphorylcholine (ChoP) into H. influenzae LPS. Expression of this gene is subject to phase variation, resulting in the spontaneous loss, or gain of phosphorylcholine (ChoP)-decorated LPS structures. To investigate the role of this phenomenon in the pathogenesis of invasive disease an H. influenzae mutant was constructed which lacked the ability to phase vary licA. This was achieved by introducing an in-frame deletion of the 5'-CAAT-3' repeats into licA using polymerase chain reaction. The resultant mutant, licADelta5'-CAAT-3', was unable to switch off expression of licA and constitutively expressed ChoP-decorated LPS structures, as judged by colony immunoblotting with Mabs 12D9 and HAS. This resulted in increased synthesis of high molecular mass LPS structures and the absence of non-ChoP-decorated LPS species as determined by T-SDS-PAGE analysis. Inability to switch off the expression of licA reduced the virulence of H. influenzae in an infant rat model of invasive disease and resulted in increased sensitivity to the bactericidal activity of serum in the presence of CRP. The ability to switch off the expression of licA through phase variation is therefore concluded to enhance the systemic survival of H. influenzae.     

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 extended lipopolysaccharide glycoforms containing two globotriose units in Haemophilus influenzae serotype b strain RM7004

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.     

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.     

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

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.     

The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.     

Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis

It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.     

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis

The availability of the complete 1.83-megabase-pair sequence of the Haemophilus influenzae strain Rd genome has facilitated significant progress in investigating the biology of H. influenzae lipopolysaccharide (LPS), a major virulence determinant of this human pathogen. By searching the H. influenzae genomic database, with sequences of known LPS biosynthetic genes from other organisms, we identified and then cloned 25 candidate LPS genes. Construction of mutant strains and characterization of the LPS by reactivity with monoclonal antibodies, PAGE fractionation patterns and electrospray mass spectrometry comparative analysis have confirmed a potential role in LPS biosynthesis for the majority of these candidate genes. Virulence studies in the infant rat have allowed us to estimate the minimal LPS structure required for intravascular dissemination. This study is one of the first to demonstrate the rapidity, economy and completeness with which novel biological information can be accessed once the complete genome sequence of an organism is available.