Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae.

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as by ESI-MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-[beta-d-Glcxp-(1-->4)]-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-d-Glcp residue was either substituted by PCho or the disaccharide branch beta-d-Galp-(1-->4)-d-alpha-d-Hepp, while the O-4 position was substituted by the globotetraose unit, beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer-core region of H. influenzae LPS. Additionally, a PEtn group was identified at O-3 of the distal heptose residue in the inner-core.     

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.     

Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae.

Twenty-three highly antibiotic-resistant strains of Haemophilus influenzae and two of Haemophilus parainfluenzae without detectable large plasmids were examined for conjugative transfer of their resistance to H. influenzae strain Rd or to other strains. Very inefficient transfer was observed for 18 H. influenzae strains and 1 H. parainfluenzae strain. All H. influenzae transcipients carried a large plasmid, and they were in turn efficient donors of their resistances in standard conjugation crosses with isogenic recipients. This was not seen for the H. parainfluenzae transcipients. It is concluded that most of the original antibiotic-resistant cultures carried an integrated conjugative R plasmid which had been excised in a few cells in each population. It was these cells which transferred resistance in the primary crosses.     

Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: comparative study with H. influenzae serotype d, strain KW20

In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NP's genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.     

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.     

Survival of nontypeable Haemophilus influenzae in macrophages

In this study we have investigated the ability of nonencapsulated, nontypeable Haemophilus influenzae, NT477 to survive in the J774 mouse macrophage-like cell line. Viable, intracellular nontypeable H. influenzae could still be recovered from macrophages 72 h after phagocytosis. In contrast, H. influenzae strain Rd, an avirulent, nonencapsulated variant of a serotype d strain, was killed within 24 h. These differences suggest that NT477, in comparison to Rd, possesses unique attributes that enable it to survive in macrophages for prolonged periods. To determine whether this trait is ubiquitous amongst nontypeable H. influenzae, 33 primary clinical isolates obtained from children with otitis media were screened for their ability to survive in macrophages. Of these isolates, 82% were able to persist in an intracellular environment for periods of at least 24 h. The number of viable organisms recovered at this time ranged from 2x10(4) to 50 colony-forming units per strain indicating that the extent to which nontypeable H. influenzae can resist macrophage-mediated killing varies between strains.     

Anomalous but helpful findings from the BBL Crystal ID kit with Haemophilus spp

Fourteen strains of Haemophilus (12 H. influenzae , 1 H. parainfluenzae and 1 H. aphrophilus ) were processed in BBL Crystal ID Enteric/Nonfermenter, API 20E and API 20NE kits, to determine whether the BBL kit misidentifies, as API kits may do, Haemophilus spp. as Pasteurella spp. The 13 H. influenzae and H. parainfluenzae strains produced uninterpretable colour reactions in the Crystal kit, thus signalling that an inappropriate species had been tested. On the other hand, the API kits (especially 20NE) often confidently 'identified' Haemophilus spp. as Pasteurella spp., giving no warning that this was a misidentification.     

Naturally occurring NAD-independent Haemophilus parainfluenzae

Four, NAD-independent, clinical isolates of Haemophilus parainfluenzae were recovered from a genital ulcer, a purulent skin lesion, a sputum specimen and a throat swab respectively. With the exception of NAD requirement, the strains exhibited the biochemical characteristics of H. parainfluenzae biotype II. The genetic relationship between these isolates and a standard strain of H. parainfluenzae was determined by testing transforming activities of two chromosomal markers, streptomycin resistance and nalidixic acid resistance. The clinical isolates were efficient donors and recipients in transformation. In addition, we demonstrated transfer of the genes conferring NAD independence to typical, NAD-requiring H. parainfluenzae and Haemophilus influenzae strains.     

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.     

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.     

Haemophilus influenzae uses the surface protein E to acquire human plasminogen and to evade innate immunity

Pathogenic microbes acquire the human plasma protein plasminogen to their surface. In this article, we characterize binding of this important coagulation regulator to the respiratory pathogen nontypeable Haemophilus influenzae and identify the Haemophilus surface protein E (PE) as a new plasminogen-binding protein. Plasminogen binds dose dependently to intact bacteria and to purified PE. The plasminogen-PE interaction is mediated by lysine residues and is also affected by ionic strength. The H. influenzae PE knockout strain (nontypeable H. influenzae 3655Δpe) bound plasminogen with ～65% lower intensity as compared with the wild-type, PE-expressing strain. In addition, PE expressed ectopically on the surface of Escherichia coli also bound plasminogen. Plasminogen, either attached to intact H. influenzae or bound to PE, was accessible for urokinase plasminogen activator. The converted active plasmin cleaved the synthetic substrate S-2251, and the natural substrates fibrinogen and C3b. Using synthetic peptides that cover the complete sequence of the PE protein, the major plasminogen-binding region was localized to a linear 28-aa-long N-terminal peptide, which represents aa 41-68. PE binds plasminogen and also vitronectin, and the two human plasma proteins compete for PE binding. Thus, PE is a major plasminogen-binding protein of the Gram-negative bacterium H. influenzae, and when converted to plasmin, PE-bound plasmin aids in immune evasion and contributes to bacterial virulence.     

Specificity in Deoxyribonucleic Acid Uptake by Transformable Haemophilus influenzae

Cells of Haemophilus influenzae strain Rd competent for genetic transformation irreversibly bound approximately five molecular fragments of H. influenzae deoxyribonucleic acid (DNA) per cell; under identical conditions, DNA derived from Escherichia coli B was not taken up (<1 molecule per 50 cells). Similarly, DNA from Xenopus laevis was not taken up by competent H. influenzae. Of the heterologous DNAs tested, only DNA from H. parainfluenzae interfered with the uptake of H. influenzae DNA, as judged by competition experiments employing either DNA binding or genetic transformation as the test system. The extracellular heterologous DNA did not suffer either single- or double-strand breakage upon exposure to competent H. influenzae.     

Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus influenzae. Identification of L-glycerol-D-manno-heptose in the outer-core region in three clinical isolates.

Structural elucidation of the lipopolysaccharide (LPS) from three nontypeable Haemophilus influenzae clinical isolates, 1209, 1207 and 1233 was achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as ESI-MS(n) on permethylated dephosphorylated OS. It was found that the organisms expressed a tremendous heterogeneous glycoform mixture resulting from the variable length of the OS chains attached to 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-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-D-Glcp residue could either be occupied by PCho or L-glycero-D-manno-heptose (L,D-Hep), which is a location for L,D-Hep that has not been seen previously in H. influenzae LPS. The outer-core L,D-Hep residue was further chain elongated at the O-6 position by the structural element beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp, or sequentially truncated versions thereof. The distal heptose residue in the inner-core was found to be chain elongated at O-2 by the globotetraose unit, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp, or sequentially truncated versions thereof. Investigation of LPS from an lpsA mutant of isolate 1233 and a lic1 mutant of isolate 1209 was also performed, which aside from confirming the functions of the gene products, simplified elucidation of the OS extending from the proximal heptose (the lpsA mutant), and showed that the organism exclusively expresses LPS glycoforms comprising the outer-core l,d-Hep residue when PCho is not expressed (the lic1 mutant).     

Mechanism of Haemophilus influenzae transfection by single and double prophage deoxyribonucleic acid.

Whole phages HP1 and HP3, vegetative-phage deoxyribonucleic acid (DNA), and single and tandem double prophage DNA were exposed to ultraviolet radiation and then assayed on a wild-type (DNA repair-proficient) Haemophilus influenzae Rd strain and on a repair-deficient uvr-1 strain. Host cell reactivation (DNA repair) was observed for whole-phage and vegetative-phage DNA but not for single and double prophage DNA. Competent (phage-resistant) Haemophilus parainfluenzae cells were normally transfected with H. influenzae-grown phage DNA and with tandem double prophage DNA but not at all with single prophage DNA. CaCl2-treated H. influenzae suspensions could be transfected with vegetative phage DNA and with double prophage DNA but not with single prophage DNA. These observations support the hypothesis that transfection with single prophage DNA occurs through prophage DNA single-strand insertion into the recipient chromosome (at the bacterial att site) followed by DNA replication and then prophage induction.     

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.     

Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118).

A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)-]-L-alpha-D-Hepp-(1-->5)-alpha-Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D (1)H-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes involved in the addition of each glycose residue.     

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.     

Characterization of chimeric lipopolysaccharides from Escherichia coli strain JM109 transformed with lipooligosaccharide synthesis genes (lsg) from Haemophilus influenzae

Previously, we reported the expression of chimeric lipopolysaccharides (LPS) in Escherichia coli strain JM109 (a K-12 strain) transformed with plasmids containing Haemophilus influenzae lipooligosaccharide synthesis genes (lsg) (Abu Kwaik, Y., McLaughlin, R. E., Apicella, M. A., and Spinola, S. M. (1991) Mol. Microbiol. 5, 2475-2480). In this current study, we have analyzed the O-deacylated LPS and free oligosaccharides from three transformants (designated pGEMLOS-4, pGEMLOS-5, and pGEMLOS-7) by matrix-assisted laser desorption ionization, electrospray ionization, and tandem mass spectrometry techniques, along with composition and linkage analyses. These data show that the chimeric LPS consist of the complete E. coli LPS core structure glycosylated on the 7-position of the non-reducing terminal branch heptose with oligosaccharides from H. influenzae. In pGEMLOS-7, the disaccharide Gal1--> 3GlcNAc1--> is added, and in pGEMLOS-5, the structure is extended to Gal1-->4GlcNAc1-->3Gal1-->3GlcNAc1-->. PGEMLOS-5 LPS reacts positively with monoclonal antibody 3F11, an antibody that recognizes the terminal disaccharide of lacto-N-neotetraose. In pGEMLOS-4 LPS, the 3F11 epitope is apparently blocked by glycosylation on the 6-position of the terminal Gal with either Gal or GlcNAc. The biosynthesis of these chimeric LPS was found to be dependent on a functional wecA (formerly rfe) gene in E. coli. By using this carbohydrate expression system, we have been able to examine the functions of the lsg genes independent of the effects of other endogenous Haemophilus genes and expressed proteins.