Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd-/b+. Description of a novel deep-rough chemotype

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd-/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a beta-(1',6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4' of the non-reducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2,2' and 3,3', respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583-587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-liquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6' of the non-reducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H-NMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: (Formula; see text)     

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I-69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd⁻/b⁺ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de- O -acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de- O -acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy- d - manno -octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS–PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.     

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.     

A hex mutant of Haemophilus influenzae.

Summary A mutant of Haemophilus influenzae which does not discriminate between low efficiency (LE) and high efficiency (HE) markers has been isolated. The mutant does not differ wild type in its sensitivity to ultraviolet radiation, methyl methanesulfonate (MMS) mitomycin C, and nitrous acid. Spontaneous mutation frequencies for three loci studied are 10-to 30-fold higher in the mutant than in the wild type strain. Low- and high-efficiency transforming markers are equally UV-resistant when assayed on this mutant. This mutant is thus similar to the hex mutant of Streptococcus pneumoniae.     

Phase diagram of deep rough mutant lipopolysaccharide from Salmonella minnesota R595.

The structural polymorphism of deep rough mutant lipopolysaccharide--in many biological systems the most active endotoxin--from Salmonella minnesota strain R595 was investigated as function of temperature, water content, and Mg2+ concentration. Differential scanning calorimetry was used to determine the amount of bound water and the enthalpy change at the beta<==>alpha gel to liquid crystalline acyl chain melting. The onset, midtemperature Tc, and completion of the beta<==>alpha phase transition were studied with Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray diffraction was used to characterize the supramolecular three-dimensional structures in each phase state. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperature Tc lying at 30 degrees C at high water content (95%) increases with decreasing water content reaching a value of 50 degrees C at 30% water content. Concomitantly, a broadening of the transition range takes place. At still lower water content, no distinct phase transition can be observed. This behavior is even more clearly expressed in the presence of Mg2+. In the lower water concentration range (< 50%) at temperatures below 70 degrees C, only lamellar structures can be observed independent of the Mg2+ concentration. This correlates with the absence of free water. Above 50% water concentration, the supramolecular structure below 70 degrees C strongly depends on the [LPS]:[Mg2+] ratio. For large [LPS]:[Mg2+] ratios, the predominant structure is nonlamellar, for smaller [LPS]:[Mg2+] ratios there is a superposition of lamellar and nonlamellar structures. At an equimolar ratio of LPS and Mg2+ a multibilayered organization is observed. The nonlamellar structures can be assigned in various cases to structures with cubic symmetry with periodicities between 12 and 16 nm. Under all investigated conditions, a transition into the hexagonal II structure takes place between 70 and 80 degrees C. These observations are discussed in relation to the biological importance of LPS as constituent of the outer membrane of gram-negative bacteria and as potent inducer of biological effects in mammals.     

Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae.

A sialylated lacto-N-neotetraose (Sial-lNnT) structural unit was identified and structurally characterized in the lipopolysaccharide (LPS) from the genome-sequenced strain Rd [corrected] (RM118) of the human pathogen Haemophilus influenzae grown in the presence of sialic acid. A combination of molecular genetics, MS and NMR spectroscopy techniques showed that this structural unit extended from the proximal heptose residue of the inner core region of the LPS molecule. The structure of the Sial-lNnT unit was identical to that found in meningococcal LPS, but glycoforms containing truncations of the Sial-lNnT unit, comprising fewer residues than the complete oligosaccharide component, were not detected. The finding of sialylated glycoforms that were either fully extended or absent suggests a novel biosynthetic feature for adding the terminal tetrasaccharide unit of the Sial-lNnT to the glycose acceptor at the proximal inner core heptose.     

Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.     

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.     

Altered lipopolysaccharide characteristic of the I69 phenotype in Haemophilus influenzae results from mutations in a novel gene, isn.

The 169 phenotype of Haemophilus influenzae results from a mutation leading to a lipopolysaccharide molecule consisting only of lipid A and a single phosphorylated 2-keto-3-deoxyoctulosonic acid residue. In this paper we describe the identification of a gene which, when mutated, results in the 169 phenotype. We have named the gene isn. The predicted amino acid sequence of Isn is homologous to the product of the lmbN gene involved in the biosynthesis of the sugar-containing antibiotic lincomycin by Streptomyces lincolnensis. lsn is situated between two loci that are homologous to the dpp and art periplasmic permease systems in Escherichia coli. Northern (RNA) blot and primer extension analyses reveal that isn is transcribed as a monocistronic mRNA. Potential functions of Isn protein are discussed.     

Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 1003.

Structural analysis of the lipopolysaccharide (LPS) of nontypeable Haemophilus influenzae strain 1003 has been achieved by the application of high-field NMR techniques, ESI-MS, capillary electrophoresis coupled to ESI-MS, composition and linkage analyses on O-deacylated LPS and core oligosaccharide material. 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)-[PP Etn-->4]-alpha-Kdop-(2-->6)-Lipid A, in which the beta-D-Glcp residue is substituted by phosphocholine at O-6 and an acetyl group at O-4. A second acetyl group is located at O-3 of the distal heptose residue (HepIII). HepIII is chain elongated at O-2 by either a beta-D-Glcp residue (major), lactose or sialyllactose (minor, i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp), where a third minor acetylation site was identified at the glucose residue. Disialylated species were also detected. In addition, a minor substitution of ester-linked glycine at HepIII and Kdo was observed.     

Identification of a novel structural motif in the lipopolysaccharide of the galE/galK double mutant of Haemophilus influenzae strain Eagan

Defined mutants of the galactose biosynthetic (Leloir) pathway were employed to investigate lipopolysaccharide (LPS) oligosaccharide expression in Haemophilus influenzae type b strain Eagan. The structures of the low-molecular-mass LPS glycoforms from strains with mutations in the genes that encode galactose epimerase (galE) and galactose kinase (galK) were determined by NMR spectroscopy on O- and N-deacylated and dephosphorylated LPS-backbone, and O-deacylated oligosaccharide samples in conjunction with electrospray mass spectrometric, glycose and methylation analyses. The structural profile of LPS glycoforms from the galK mutant was found to be identical to that of the galactose and glucose-containing Hex5 glycoform previously identified in the parent strain [Masoud, H.; Moxon, E. R.; Martin, A.; Krajcarski, D.; Richards, J. C. Biochemistry1997, 36, 2091-2103]. LPS from the H. influenzae strain bearing mutations in both galK and galE (galE/galK double mutant) was devoid of galactose. In the double mutant, Hex3 and Hex4 glycoforms containing di- and tri-glucan side chains from the central heptose of the triheptosyl inner-core unit were identified as the major glycoforms. The triglucoside chain extension, β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, identified in the Hex4 glycoform has not been previously reported as a structural element of H. influenzae LPS. In the parent strain, it is the galactose-containing trisaccharide, β-d-Galp-(1→4)-β-d-Glcp-(1→4)-α-d-Glcp, and further extended analogues thereof, that substitute the central heptose. When grown in galactose deficient media, the galE single mutant was found to expresses the same population of LPS glycoforms as the double mutant.     

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.     

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.     

Structural analysis of the lipopolysaccharide from the nontypable Haemophilus influenzae strain SB 33.

The structure of the core region of the lipopolysaccharide (LPS) from the nontypable Haemophilus influenzae strain SB 33 was elucidated. The LPS was subjected to a variety of degradative procedures. The structures of the derived oligosaccharide products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. These analyses revealed a series of related phosphocholine (PCho) containing structures differing in the number of hexose residues. The results pointed to each species containing a conserved phosphoethanolamine (PEtn) substituted heptose-containing trisaccharide inner-core moiety. The major LPS glycoforms were identified as 2-Hex, 3-Hex and 4-Hex species according to the number of hexose residues present.     

Generation of lipooligosaccharide mutants of Haemophilus influenzae type b.

We previously reported the analysis of recombinant plasmids from Haemophilus influenzae type b (Hib) that lead to modifications of Escherichia coli lipopolysaccharide (LPS) (Y. Abu Kwaik, R. E. McLaughlin, M. A. Apicella, and S. M. Spinola, Mol. Microbiol. 5:2475-2480, 1991). The modified LPS species are recognized by monoclonal antibodies (MAbs) 6E4 and 3F11. MAb 6E4 binds to a stable 2-keto-3-deoxyoctulosonic acid epitope, while MAb 3F11 binds to a Gal beta 1-4GlcNac epitope that phase varies in Hib at a frequency of 2 to 5%. The internal EcoRI fragment containing most of the DNA required for LPS modification in E. coli was used as the target for transposon mutagenesis. Plasmids containing minitransposon m-Tn3(Cm) randomly inserted into the target fragment were transformed into the isogenic Hib strain, and transposon integration into the Hib chromosome was verified by colony hybridization. The lipooligosaccharides of 36 transformants were phenotypically and antigenically characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with a variety of MAbs that recognize both stable and phase-varying lipooligosaccharide epitopes. The majority of the mutants had altered reactivity with MAb 6E4. With one exception, these mutants retained the ability to express phase-varying epitopes. Analysis of the transformants suggested that the 6E4 epitope was contained on an oligosaccharide chain separate from that of phase-varying epitopes and appeared to be assembled in at least three separate steps.     

The dilution rate affects the outer membrane protein and lipopolysaccharide composition of Haemophilus influenzae type b grown under iron limitation.

When Haemophilus influenzae type b was grown under iron limitation in continuous culture, the dilution rate affected the outer membrane protein and lipopolysaccharide composition. Investigations of the effect of the reduced availability of iron or other environmental parameters on these surface components should be controlled for growth rate.     

Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide.

A survey of both typeable and nontypeable strains of Haemophilus influenzae indicated that they contain glycine (Gly) in their lipopolysaccharide (LPS). Significant amounts (30-250 pmol Gly/microg LPS) were determined by high-performance anion-exchange chromatography using pulsed amperometric detection after treatment of the LPS with mild alkali. Oligosaccharides obtained from LPS after mild acid hydrolysis and gel filtration chromatography were investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis (CE) ESI-MS. In all cases, molecular ions corresponding to the major glycoforms were identified and were accompanied by ions differing by 57 Da, thus indicating the presence of glycine. The position of glycine in these glycoforms was determined by CE-ESI-MS/MS analyses. It was found that, depending on strain, glycine can substitute each of the heptoses of the inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-alpha-Kdo of H. influenzae LPS as well as Kdo. In some strains, mixtures of monosubstituted Gly-containing glycoforms having different substitution patterns were identified.