Three genes, lgtF, lic2C and lpsA, have a primary role in determining the pattern of oligosaccharide extension from the inner core of Haemophilus influenzae LPS

Derek W. Hood1, Mary E. Deadman1, Andrew D. Cox2, Katherine Makepeace1, Adele Martin2, James C. Richards2 and E. Richard Moxon1

1 Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada, K1A 0R6

Correspondence
Derek W. Hood
derek.hood{at}paediatrics.ox.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lipopolysaccharide (LPS) is a virulence determinant of Haemophilus influenzae and exhibits substantial heterogeneity in structure within and between strains. Key factors contributing to this heterogeneity are the genes required to add the first glycose to each of the three heptose residues of the LPS inner core. In each case this addition can facilitate further oligosaccharide extension. lgtF is invariably present in strains and the product has a function in adding the glucose to the first heptose. lic2C is present in half the strains and was found to add a glucose to the second heptose. Insertion of lic2C into a strain that does not naturally contain it resulted in hexose incorporation from the second heptose of the LPS. The product of the lpsA gene can add a glucose or galactose to the third heptose. By allelic replacement of lpsA between strains it is shown that the sequence of the gene can be the sole determinant of this specificity. Thus, lgtF, lic2C and lpsA make significant but very distinct contributions to the conservation and variable patterns of oligosaccharide extensions seen in H. influenzae LPS.


Abbreviations: Hep, heptose; Hex, hexose; Kdo, 2-keto-3-deoxyoctulosonic acid; MS-MS, tandem mass spectroscopy; NTHi, non-typeable H. influenzae; PCho, phosphocholine; PEtn, phosphoethanolamine


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Haemophilus influenzae is an important cause of human disease. The bacterium normally colonizes the upper respiratory tract and can cause respiratory tract infections by contiguous spread. Occasionally, strains can be invasive, causing bacteraemic infections such as meningitis and septicaemia, particularly in infants. Lipopolysaccharide (LPS) is a major and essential component of the cell wall of H. influenzae and is a virulence determinant. LPS can have a role at each stage of the pathogenesis of H. influenzae infections, causes cytotoxic injury to tissues and is a target for host immune responses (reviewed by Hood & Moxon, 1999). H. influenzae LPS is a complex glycolipid composed of a membrane-anchoring lipid A portion linked by a single 2-keto-3-deoxyoctulosonic acid (Kdo) molecule to a heterogeneous oligosaccharide composed of neutral heptose (Hep) and hexose (Hex) sugars (Zamze & Moxon, 1987). Detailed analysis of LPS from a number of strains has led to a structural model whereby each Hep of a conserved trisaccharide inner core can be a point for the addition of Hex sugars and further chain extension. The degree and pattern of Hex extension varies between strains. Phosphate-containing substituents, including free phosphate groups (P), phosphoethanolamine (PEtn), pyrophosphoethanolamine (PPEtn) and phosphocholine (PCho), and glycine and O-acetyl group substituents further contribute to the structural heterogeneity of H. influenzae LPS. The conserved inner core and extremely heterogeneous outer core is a striking feature of H. influenzae LPS. Several of the surface-exposed epitopes of H. influenzae LPS are subject to high frequency phase-variation (Patrick et al., 1987; Weiser et al., 1989), an adaptive mechanism that is advantageous for survival of bacteria confronted by the differing microenvironments and immune responses of the host. This heterogeneity has complicated the structural and biological analysis of H. influenzae LPS, so the construction of mutant strains, with defined core structures, has been a priority. Our detailed analysis of LPS oligosaccharide synthesis has focussed on three strains. The type d derived strain, RM118 (Rd), produces LPS with a globotetraose [{beta}-D-galpnac-(1->3)-{alpha}-D-galp-(1->4)-{beta}-D-galp-(1->4)-{beta}-D-Glcp] extension from the third heptose (HepIII) and a single glucose as extension from the first heptose (HepI) (Fig. 1) (Risberg et al., 1999). The type b strain, RM153, has LPS containing a single glucose and a single galactose as extensions from HepI and HepIII, respectively. In the fully extended glycoform of this strain there is an {alpha}-D-galp-(1->4)-{beta}-D-galp-(1->4)-{beta}-D-glcp-(1->4)-{alpha}-D-Glcp as the extension from the second heptose (Masoud et al., 1997). The type b strain RM7004 has the same LPS structure as strain RM153 but with an {alpha}-D-galp-(1->4)-{beta}-D-galp-(1->4)-{beta}-D-glcp-(1->4)-{beta}-D-Glcp from HepI in the fully extended glycoform (Masoud et al., 2003). A comprehensive analysis of multiple LPS biosynthetic loci in the related type b strains RM153 and RM7004 has been undertaken (Hood et al., 1996a). In a later study, we identified each of the glycosyltransferases involved in the assembly of the LPS oligosaccharide in H. influenzae strain RM118 (Rd) (Hood et al., 2001a).



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Fig. 1. Schematic representation of the LPS structure of H. influenzae strains RM153 (upper) and RM118 (lower). The relevant sugars linked by the products from the genes lgtF, lic2C and lpsA are shown by arrows. Abbreviations: Kdo, 2-keto-3-deoxyoctulosonic acid; Hep, L-glycero-D-manno-heptose; Glc, D-glucose; Gal, D-galactose; GalNAc, N-acetylgalactosamine; PEtn, phosphoethanolamine; P, phosphate; PC, phosphocholine. For each LPS structure the heptose backbone comprises from top to bottom HepI, HepII and HepIII. Non-stoichiometric substitutions are shown by dotted lines. The type b strain RM7004 has the same LPS structure as strain RM153 but with an {alpha}-D-galp-(1->4)-{beta}-D-galp-(1->4)-{beta}-D-glcp-(1->4)-{beta}-D-Glcp from HepI in the fully extended glycoform (Masoud et al., 2003).

 
In this manuscript we investigate three key genes, lgtF, lic2C and lpsA, that function to add the initial sugars of the oligosaccharide extensions from the inner core of H. influenzae LPS. These additions are a prerequisite in each case for any further hexose extension from the inner core, with the potential to influence both the biology and the virulence of the organism.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
H. influenzae strain RM118 was from the same source as strain Rd used for the genome sequencing project (Fleischmann et al., 1995). The type b strains RM153 (Eagan) and RM7004 are both disease isolates from USA and Holland, respectively. A representative group of 28 H. influenzae capsulate strains, including all capsular types, and a group of 25 non-typeable (NTHi) strains described previously (Hood et al., 1999) were used for genotype analysis. Strains were grown at 37 °C in brain heart infusion (BHI) broth supplemented with haemin (10 µg ml–1) and NAD (2 µg ml–1) or on BHI agar supplemented with 10 % Levinthals reagent. For selection following transformation, kanamycin (10 µg ml–1) was added to the growth medium.

Escherichia coli strain DH5{alpha} was grown at 37 °C in Luria–Bertani (LB) broth supplemented with ampicillin (100 µg ml–1) or kanamycin (50 µg ml–1) as required (Sambrook et al., 1989).

PCR amplification and recombinant DNA methodology.
Restriction endonucleases and DNA modifying enzymes were obtained from Roche and used according to the supplier's instructions. Plasmid DNA preparation, Southern blotting and hybridization were performed as described by Sambrook et al. (1989). Chromosomal DNA was prepared from Haemophilus by the method described previously (Hood et al., 1996a).

Plasmids containing cloned and interrupted lgtF and lpsA genes have been described previously (Hood et al., 2001a, 1996a). lic2C, previously called lic2orf3 (GenBank U36398; High et al., 1996), was amplified by PCR with the adjacent reading frame, lic2B, from chromosomal DNA of strain RM153 using the primers lic2BA (5'-CAATTTAGCGATGAGTTCC-3') and lic2BB (5'-AAGTATGATCCTCAAATG-3'). PCR conditions were for 1 min periods of denaturation (94 °C), annealing (50 °C) and polymerization (72 °C) for 30 cycles. One microlitre of PCR product was ligated with 50 ng plasmid pT7Blue (Novagen) and transformed into E. coli. Recombinant plasmids were confirmed by restriction endonuclease digestion and sequencing from plasmid-specific primers. The lic2C gene was inactivated by inserting a kanamycin resistance cassette (released by digestion with HincII from pUC4K; Amersham Biotech) into an EcoRV restriction site located towards the 5' end of lic2C to give plasmid pCK4, used for construction of lic2C mutant strains. Oligonucleotide primers L2E (5'-TTTCGCCATTGTATCCTC-3'), designed against sequence at the 5' end of HI0548 (infA), and lic2BA were used under the same PCR conditions described above to investigate the chromosomal location of lic2C in type b strains.

For the transfer of lpsA genes between strains, a region of genomic DNA was amplified from strains RM118 and RM153 by PCR using primers 6018F (5'-TACTTAGCTCGCATTCTC-3') and 6018G (5'-AACACTCAAATGCTCATC-3') under the conditions described above. The 2·6 kb fragment comprising lpsA (HI0765), the flanking reading frames HI0764 and HI0766, and segments of HI0763 and HI0767, was cloned into pT7Blue as described above. Confirmed clones were designated p10.6 (RM118) and p11.7 (RM153). HI0767 was inactivated in each clone by digestion with restriction endonuclease SnaBI then ligation with a kanamycin resistance cassette (released by digestion with HincII from pUC4Kan) to give plasmids p11.7S and p10.7S for strains RM153 and RM118, respectively.

The presence or absence of LPS biosynthesis genes was investigated in H. influenzae strains using the following primer pairs: lgtFa (5'-TGGTGGTGGGCAAGACGC-3') and lgtFb (5'-AGCCTGAATTCGACAGCC-3') for lgtF; lic2BA and lic2BC (5'-CAATTTCACTAACTTGCC-3') for lic2C; and 6018A (5'-GCGTGGCGACAATTAGGC-3') and 6018C (5'-TTGAATATCGTTTAGCAC-3') for lpsA. PCR amplification was carried out on chromosomal DNA under the conditions described above.

Construction of mutant strains.
H. influenzae strains mutated in lpsA and strain RM118 mutated in the lgtF gene have been reported previously (Hood et al., 1996a, 2001a). To make the RM153 and RM7004 lgtF and lic2C mutants and the lpsA heterologous gene replacements, 2–3 µg of the appropriate linearized plasmid DNA construct was used to transform H. influenzae (Herriott et al., 1970) and transformants were selected upon kanamycin. To construct strain RM118lic2Alic2B+lic2C+, RM118 was transformed with 5 µg chromosomal DNA isolated from a RM153lic2A mutant (D. Hood, unpublished). All transformants were checked by reculturing on BHI/kanamycin, then confirmed as mutants by PCR amplification and Southern analysis of endonuclease-digested chromosomal DNA. For the strains transformed with the heterologous lpsA genes, correct transformants were confirmed by obtaining the DNA sequence of the lpsA gene using cycle sequencing and primers designed against the Rd genome sequence. Sequencing reactions were carried out using ABI Big Dye Sequencing reagents and were analysed on an ABI377 autosequencer.

Analysis of LPS by immunoblotting and electrophoresis.
The reactivity of H. influenzae strains to LPS-specific mAbs and the patterns of LPS isolated from these strains upon Tricine-SDS-PAGE (T-SDS-PAGE), were analysed as described previously (Hood et al., 1996a).

Serum resistance assay.
Bacteria were assayed for survival against the killing effect of normal human serum in three separate experiments as described previously (Hood et al., 1999).

Structural analysis of LPS.
Cells from 10 l batch cultures (10 lots of 1 l) were harvested after overnight growth, LPS was extracted by the hot phenol/water method (Cox et al., 2002) followed by ethanol precipitation as described by Thibault & Richards (2000). LPS was purified by repeated ultracentrifugation (105 000 g, 4 °C, 2x 5 h) and samples were analysed as their O-deacylated derivatives (LPS-OH) using MS and NMR techniques as described previously (Cox et al., 2002; Hood et al., 2001a).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Occurrence and function of lgtF, lic2C and lpsA
The glycosyltransferase LgtF in H. influenzae strain RM153 was found to add the {beta}-D-Glcp to HepI of the LPS triheptose backbone, corroborating the function found previously in strain RM118 (Hood et al., 2001a). Tandem MS (MS-MS) analysis of the LPS from an lgtF mutant constructed for strain RM153 revealed a structure comprising the triheptose backbone and only a single Hex residue (Table 1), shown to be galactose by sugar analysis. NMR and methylation analyses indicated this galactose to be located as the sole extension from the third heptose as seen in wild-type RM153 LPS. In the truncated LPS from RM153lgtF (Fig. 2), loss of the glucose from HepI thus prevented any oligosaccharide extension from HepII, -{alpha}-D-galp-(1->4)-{beta}-D-galp-(1->4)-{beta}-D-glcp-(1->4)-{alpha}-D-Glcp-(1->3)- in its full form, seen in the LPS of the parent strain.


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Table 1. Negative ion electrospray MS (ES-MS) data and proposed composition of O-deacylated LPS from H. influenzae strains RM118 and RM153 mutants

Mean mass units were used for calculation of molecular mass based on proposed composition as follows: Lipid A, 953·00; Hex, 162·15; HexNAc, 203·19; Hep, 192·17; Kdo-P, 300·16; PEtn, 123·05. PCho, 165·05; sialic acid (Sial), 291·00.

 


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Fig. 2. The migration patterns of LPS from strains RM118 and RM153 and derived strains with mutations in glycosyltransferase genes or containing heterologous lpsA genes after T-SDS-PAGE and staining with silver. The mutants are listed by the relevant LPS gene; lpsAe and lpsAr are the gene replacements from RM153 and RM118, respectively; wt, wild-type. The arrow on the left indicates the position of the 6·5 kDa protein marker when run on the same gel. The lower intense-staining bands in each profile represent the major glycoform(s). Other bands represent alternative minor glycoforms produced mainly through phase-variable expression of LPS biosynthesis genes.

 
The lgtF gene was universally present in the 28 capsulate and 25 NTHi strains tested (data not shown). Reactivity of a panel of mAbs with the lgtF mutant strains showed greatly reduced binding for strain RM118lgtF, compared to wild-type, for mAb TEPC-15. mAb TEPC-15 reacts with PCho, which in strain RM118 is attached to the glucose extended from HepI (Fig. 1). For strain RM118lgtF, which lacks this extension, there is correspondingly no PCho addition. In strain RM153, PCho is added to the galactose extended from HepIII in a minor proportion of glycoforms (data not shown).

The lic2C gene was cloned, mutated, then used to make a lic2C mutant in strain RM153. The lic2 locus has been investigated in previous studies of LPS synthesis in this laboratory, but the function of the third reading frame, lic2orf3 now renamed lic2C (Fig. 3), was not elucidated (High et al., 1993, 1996). PAGE analysis indicated that LPS from strain RM153lic2C was truncated when compared to LPS from the parent strain (Fig. 2). MS-MS analysis of LPS from strain RM153lic2C indicated that the major species was a Hex2 glycoform (Table 1). NMR and methylation analyses indicated that this glycoform contained a single glucose and a single galactose as sole extensions from HepI and HepIII, respectively. No extension was found from HepII, thus indicating that a likely function of Lic2C was in the addition of the first hexose, {alpha}-D-Glcp, to the 3-position of HepII. Strain RM153lic2C no longer reacted with mAb 4C4 (data not shown). mAb 4C4 reacts with a digalactoside epitope of H. influenzae LPS (Virji et al., 1990), which in strain RM153 is found as the third and fourth sugars in the extension from HepII. lic2C has some homology to a gene, lgtG from Neisseria species, encoding a glucosyltransferase adding the glucose residue to the 3-position of the corresponding heptose of Neisseria LPS (Banerjee et al., 1998).



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Fig. 3. Organization of chromosomal regions, including the lic2 and lpsA genes in H. influenzae. (a) The lic2 locus in the type b strain RM7004 and strain RM118 (Rd); (b) lpsA and surrounding genes in strain RM118. Genes and their direction of transcription are represented by the arrows. In (a) the locations of oligonucleotides are shown by small arrows below the genes. The relative positions of the flanking genes, apaH and infA, adjacent to the lic2 locus are shown by the dotted lines. In (b) the point for insertion of the kanamycin resistance cassette in HI0767 is indicated by a solid vertical line and the extent of the region cloned from each strain is shown by the vertical dashed lines.

 
lic2C and the adjacent gene, lic2B, are absent from our strain RM118 and the Rd genome sequence; this strain has previously been shown to lack any oligosaccharide extension from HepII in its LPS (Risberg et al., 1999). In the genome sequence, the adjacent reading frame to lic2A (HI0550) and ksgA (HI0549), which are present, is HI0548 (translation initiation factor 1 gene, infA) (Fig. 3). The DNA sequence adjacent to the lic2 locus in the type b strains is not known. Utilizing an oligonucleotide primer designed against the 5' sequence of HI0548 in combination with primer lic2BA, a PCR product of 2·1 kb was amplified from strains RM153 and RM7004 (data not shown), indicating that in these strains lic2B and lic2C are simply inserted between ksgA and HI0548. lic2C was investigated by PCR amplification in 27 strains and was found to be present in only 13 of these strains tested.

To confirm the function of lic2C, we transformed strain RM118 with chromosomal DNA from a RM153lic2A mutant strain in an attempt to introduce a functional lic2C gene into the genome of that strain. This DNA has a kanamycin resistance gene in lic2A that can act as a selectable marker for transformation. Transformation with chromosomal DNA in H. influenzae typically results in multiple gene integration from the donor into the recipient. We screened kanamycin-resistant transformants of strain RM118 by PCR for the presence of the lic2C and lic2B genes and found these at high frequency. LPS prepared from the transformed strain, RM118lic2Alic2C+lic2B+, had some higher molecular mass species upon PAGE analysis than LPS from the corresponding lic2A mutant, where lic2C and lic2B are absent (Fig. 2). MS-MS analysis of the LPS indicated that the major species produced by this strain were Hex2 and Hex3 glycoforms (Table 1). LPS from a RM118lic2A mutant has been previously shown to comprise only Hex2 glycoforms (Hood et al., 2001a), with a single glucose as the extension from both HepI and HepIII of the inner core. NMR and methylation analyses of RM118lic2Alic2C+lic2B+-derived LPS indicated that the Hex3 glycoform contained a glucose residue as extension from HepII. This was shown by the anomeric proton resonance of the {alpha}-Glc residue at 5·30 p.p.m. (Fig. 4) that in a NOESY experiment (data not shown) was found to substitute the HepII residue at the 3-position. This strain contains active lic2C and lic2B genes. To provide further evidence that lic2C specifically was responsible for addition of the glucose, SDS-PAGE analysis of LPS from strain RM118 transformed with chromosomal DNA from strain RM153lic2B also showed an increase in molecular mass when compared to wild-type (data not shown). In this strain, RM118lic2A+lic2Blic2C+, the lic2B gene is inactivated, but there remains a complete lic2C gene. This supports our conclusion that Lic2C is the transferase responsible for initiating chain extension from HepII of H. influenzae LPS.



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Fig. 4. Low-field anomeric region of the 1H-NMR spectrum for O-deacylated LPS from H. influenzae strain RM118lic2Alic2B+lic2C+. The spectrum was recorded in D2O at 25 °C. Splitting of the HepII anomeric 1H resonances are due to non-stoichiometric substitution of HepII with the {alpha}-Glc.

 
The lpsA gene was universally present in the set of H. influenzae strains tested in this study. The structure of LPS derived from lpsA mutants of strain RM153 (Hood et al., 1996a) and RM118 (Hood et al., 2001a) has been investigated previously. The lpsA gene is required to add the first hexose to HepIII of the LPS, this being glucose in RM118 and galactose in RM153. To confirm our prediction that the specificity of LpsA function is defined by the DNA sequence of any particular lpsA gene, we amplified by PCR, then cloned a DNA fragment containing lpsA, the flanking genes (termed HI0764 and HI0766 in the Rd genome sequence) and part of each of HI0763 and HI0767 from strains RM118 and RM153. For selection following transformation, a kanamycin resistance cassette was inserted into the cloned DNA flanking the lpsA gene. HI0764 encodes an essential gene product (RibB) and HI0766 contained no suitable restriction enzyme sites. Thus, HI0767, encoding a conserved hypothetical protein of unknown function, was disrupted in the plasmid constructs. Following transformation into H. influenzae, the exchange of the complete heterologous lpsA gene in transformants by recombination was confirmed by DNA sequence analysis. The strains were named RM118(lpsAe), RM153(lpsAr) and RM7004(lpsAr), where lpsAe originates from the RM153 clone (p11.7S) and lpsAr from the RM118 clone (p10.6S). Transformants containing the homologous genes, RM118(lpsAr) and RM153(lpsAe) and strain RM7004(lpsAe) containing the gene from strain RM153 were also confirmed. PAGE analysis showed changes in the electrophoretic pattern of LPS from strains containing a heterologous lpsA gene when compared to the relevant wild-type (Fig. 2). Strains transformed with the homologous genes showed little change, albeit that the proportions of glycoforms expressed by strain RM118(lpsAr) are somewhat altered when compared to the wild-type profile in Fig. 2. It is unlikely that HI0767 has any role associated with LPS synthesis. LPS from strain RM153(lpsAr) showed an increase in molecular mass when compared to that from RM153, whilst LPS from strain RM118(lpsAe) showed a reduction in molecular mass when compared to RM118 (Fig. 2).

A panel of mAbs was used to assess changes in the LPS of strains expressing the heterologous lpsA genes. mAb L6A9 showed changes in reactivity between the isogenic strains (data not shown). An important element of the LPS epitope required for mAb L6A9 to bind has been shown to be the hexose added directly to HepIII, being optimal when a galactose is present (M. Gidney, unpublished). The reactivity of strain RM118(lpsAe) increased when compared to RM118, consistent with truncation of the extension from HepIII due to exchange of the proximal glucose for a galactose residue. Strain RM153(lpsAr) showed some reduction in mAb L6A9 binding when compared to wild-type, consistent with exchange of the galactose from HepIII with either a glucose or no sugar being added. Strains RM118 and RM153 with mutations in the lpsA gene both showed reduced binding of mAb L6A9 when compared to wild-type (data not shown).

The structure of LPS purified from strains RM118(lpsAe) and RM153(lpsAr) was analysed by MS-MS and capillary electrophoresis MS (CE-MS). RM153(lpsAr) showed a distribution of glycoforms from Hex4 to Hex6 (Table 1) that is similar to that found in wild-type LPS preparations (Masoud et al., 1997) (Fig. 1). The Hex4 glycoforms in this strain were found to include no hexose extension from HepIII, indicating that the lpsA gene from strain RM118 is less able to incorporate a hexose in the RM153 LPS structure than is the native gene. Strain RM118(lpsAe)-derived LPS contained a predominance of Hex2 glycoforms and small amounts of Hex1 and Hex3 forms (Table 1), and was more truncated than that from wild-type (Risberg et al., 1999) (Fig. 1). The Hex1 glycoform is likely to be that containing only a glucose attached to HepI in the absence of any extension from HepIII (Hood et al., 2001a). The Hex2 glycoform contains a hexose substitution at the 2-position of HepIII. Sugar analysis suggested this hexose to be a galactose, an observation confirmed by NMR (data not shown). The nature of minor glycoforms in this strain remains to be investigated. Thus, in both strains the expression of the heterologous lpsA gene resulted in an altered LPS structure. In strain RM118, lpsAe or lpsAr directs incorporation of the gene-type specific hexose to HepIII, being a galactose and glucose, respectively.

Obtaining DNA sequence of the gene from the type b strains RM153 and RM7004 and comparing these with the sequence in the Rd genome database, equivalent to the gene from our strain RM118, investigated the basis for the heterogeneity of function observed for LpsA. Over the 849 bp ORF there were 15 nucleotide polymorphisms (1·8 % of sequence) between the sequences (data not shown). For a majority of these (12/15) the sequences were the same for the two type b strains and different for Rd, but for some (3/15) the Rd sequence matched that of one of the type b strains. Six of these 15 polymorphisms would lead to changes in the translated amino acid sequence.

Serum resistance
The biological relevance of oligosaccharide chain extension in H. influenzae LPS facilitated by the genes lgtF, lic2C and lpsA was investigated using a serum resistance assay. The killing effect of pooled human sera on mutants in strains RM153, RM118, RM7004 and a representative NTHi strain, 375, were assayed and compared to wild-type. For strain RM118, mutations in both the lgtF and lpsA genes led to the most significant reduction in the level of bacterial resistance to killing by normal human serum (Fig. 5). Typically, the percentage of serum required to kill 60 % of wild-type RM118 organisms was 2·5 %, whereas an lgtF or lpsA mutant was killed to the same level by less than 0·6 % serum. This relates to the loss of the globotetraose from the LPS of strain RM118lpsA and primarily the glucose and PCho attached to HepI for RM118lgtF. A RM118lic1 mutant strain lacking PCho (Hood et al., 2001a) behaved as wild-type in the assay (data not shown). A similar reduction in serum resistance was observed for strain RM118(lpsAe) (Fig. 5). Strain RM118lic2Alic2B+lic2C+ was more resistant to killing than strain RM118lic2A. For strain RM153, the level of serum required to kill 60 % of wild-type organisms was increased, as would be expected for a capsule-expressing strain, to around 20 % pooled human serum in this experiment (Fig. 5). Only very minor changes were seen for RM153lpsA or RM153lic2C strains, but again a significant change was observed for the lgtF mutant expressing the most truncated LPS (Fig. 5). Sixty percent of RM153lgtF bacteria were killed in the presence of 0·25 % human serum, whereas strain RM153lic2C that expresses LPS containing only predominantly one sugar extra is more equivalent to wild-type. Again, loss of the glucose from HepI of the LPS precipitated the dramatic reduction observed in serum resistance. Strain RM153(lpsAr), expressing the heterologous lpsA gene, demonstrated a level of serum killing very similar to wild-type (Fig. 5). In the type b strain, RM7004, mutation of lgtF showed a similar effect to that seen with strain RM153, but in this strain mutation of lpsA and lic2C also resulted in significant reduction to killing by normal human serum when compared to wild-type (Fig. 5). This might reflect the more extensive oligosaccharide extensions observed in the LPS of strain RM7004. For a representative NTHi strain, 375, mutation of lpsA resulted in no change from the wild-type level of killing, but again an lgtF mutant strain showed a significantly increased susceptibility to killing by normal human serum (data not shown).



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Fig. 5. Resistance of H. influenzae strains to the killing effect of normal human serum. (a) Strain RM118 and derived mutants; (b) strain RM153 and derived mutants; (c) strain RM7004 and derived mutants. lic2orf3 represents lic2C. Organisms (5x102) were added to doubling dilutions of serum; results are expressed as percentage survival of inoculating bacteria against serum concentration. Each experiment was repeated three times and showed consistent differences between strains. Representative profiles from the same experiment are illustrated.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Structural analyses of LPS from a number of strains of H. influenzae have indicated that the molecule can be considered as comprising two contrasting parts. There exists a triheptose inner core backbone that is conserved between strains from which extend oligosaccharides of the outer core that are very heterogeneous between strains. The lgtF, lic2C and lpsA genes encode glycosyltransferase enzymes responsible for adding the first glycose to HepI, HepII and HepIII, respectively, of the LPS inner core and have key, but different, roles in determining the patterns of oligosaccharide extension observed in the LPS across H. influenzae strains. An understanding of LPS heterogeneity is crucial to our aims to comprehend the role of LPS and its structural variation in bacterial virulence and to test the molecule as a vaccine candidate to prevent H. influenzae disease.

The glucose added to HepI by LgtF is conserved in the LPS of all H. influenzae strains studied to date and indeed it might be considered that this sugar is part of a conserved glucose-triheptose-containing common inner core. For strains such as RM7004, which elaborate further chain extension from HepI beyond the glucose, mutation of lgtF prevents all such addition. LgtF has homology to LgtF (IcsB) in the pathogenic Neisseria species where it carries out a related function, adding glucose as the first sugar in the extension from the proximal heptose of the LPS inner core.

The glycosyltransferases LgtF, Lic2C and LpsA, appear to have different degrees of dependence upon other oligosaccharide extensions for their function in a number of strains tested. The processes of hexose extension from both HepI and HepIII appear to be largely independent of other chain extensions, unlike that from HepII. In strain RM153, a mutation in lgtF results in no chain extension from HepII. An RM153lic2C strain produces LPS with no extension from HepII, but retains the single hexose extensions from HepI and HepIII. Thus, at least in strain RM153, extension from the second heptose would appear to require the addition of the glucose to HepI for it to proceed. A consistent feature of H. influenzae LPS is that for oligosaccharide chains to be extended past the first hexose added to each heptose of the inner core backbone, a glucose is required as the first sugar.

The lic2C gene is present in only about half of the H. influenzae strains tested and its distribution correlates with oligosaccharide extension from HepII in strains where details of the LPS structure are known. LPS from strains RM153 (Masoud et al., 1997), RM7004 (Masoud et al., 2003) and NTHi 486 (Månsson et al., 2001) have such an extension and lic2C is present. Strains RM118 (Risberg et al., 1999) and NTHi 375 (Hood et al., 1999), 1003 (Månsson et al., 2002), 1207, 1209 and 1233 (Månsson et al., 2003) have no such chain extension and lack the gene. Thus, the presence of lic2C determines one aspect of H. influenzae LPS complexity, namely, oligosaccharide chain extension from HepII. The lic2 locus was investigated previously in our laboratory and was described as comprising genes in the order of lic2A, ksgA, lic2orf3 (now renamed lic2C) and lic2B (Fig. 3) (High et al., 1993, 1996). lic2A is a phase-variable gene, encoding a {beta}-galactosyltransferase required for the biosynthesis of a digalactoside epitope of H. influenzae LPS (Hood et al., 2001a), but the functions of lic2orf3 and lic2B were not elucidated in the original studies. However, in a subsequent structural study of LPS from a strain derived from RM7004 by deleting the entire lic2 locus, the glycoforms were found to be more truncated than those from the isogenic lic2A mutant (Schweda et al., 2000). Lic2C has over 60 % sequence similarity to LgtG, an enzyme required for addition of a glucose to the second heptose in the LPS of pathogenic Neisseria species (Banerjee et al., 1998).

The LpsA enzyme from H. influenzae appears to be somewhat unique amongst bacterial glycosyltransferases. The transferase encoded by the same gene can add a glucose (RM118) or a galactose (RM153) linked to the 2-position of HepIII. The DNA sequence of lpsA from two type b strains and strain Rd is highly conserved, yet reciprocal replacement of the gene between strains indicates that the few sequence changes that do exist are all that is required to direct the addition of a glucose or a galactose. The lpsA gene has homology to other LPS-related genes, including the lpsA gene from Mannheimia (Pasteurella) haemolytica, the lic2A and lic2B genes of H. influenzae and the lgtB and lgtE genes from the pathogenic Neisseria (Gotschlich, 1994). LPS genes with related sequence are not uncommon within an organism, but such genes are generally more divergent in sequence and invariably function at different points in the LPS biosynthetic pathway. Recently, it has been shown that LpsA can add a glucose linked to the 3-position of HepIII in strain NTHi 486 (Månsson et al., 2001). The fourth possible combination of function, i.e. adding a galactose to the 3-position of HepIII, will also be likely to be found in the LPS of NTHi strains currently being studied (unpublished results). This would indicate that LpsA is indeed multifunctional in terms of the specificity of its role in LPS synthesis. In each strain studied to date, LPS contains uniquely only a glucose or galactose linked to HepIII in one of the two possible linkages and no mixture has been found. The lpsA gene defines a further mechanism contributing to the heterogeneity of LPS oligosaccharide structure in H. influenzae. Sequence analysis of the lpsA gene from strains where the detailed structure of the LPS is known, and LpsA function is known or can be predicted, should define the amino acids underlying the specificity of function. Upon completion of these analyses, the question of whether lpsA constitutes a single gene, or a gene family, and how these altered sequences have evolved, can be properly addressed.

Oligosaccharide extensions from the inner core are important for the biological function of H. influenzae LPS and the role of the molecule in virulence (Hood et al., 1996a). Part of this effect may be relatively non-specific in that changes in the total length of oligosaccharide extensions of the LPS will modulate the hydrophobic barrier around the bacterial cell. However, it may be more important that epitopes with specific biological roles are no longer incorporated into truncated LPS. Such epitopes are the digalactoside [-{alpha}-D-galp-(1->4)-{beta}-D-Galp-], PCho and sialic acid. The digalactoside which is predicted to mimic host-cell surface epitopes can form part of the oligosaccharide extension from all heptoses in H. influenzae LPS (Risberg et al., 1999; Masoud et al., 2003) and can increase intravascular survival of the organisms in an animal model (Hood et al., 1996b). PCho is a substituent of LPS and is important for susceptibility to killing by C-reactive protein-dependent mechanisms in the host and can contribute to persistence in the nasopharynx in in vivo model systems (Weiser & Pan, 1998; Weiser et al., 1998). PCho is typically attached to a hexose directly linked to any one of the three heptoses, the sugars added by LgtF, LpsA and Lic2C. The location of PCho in the LPS molecule can significantly alter the binding properties of the moiety and hence the resistance properties of the bacterium to host defence mechanisms (Lysenko et al., 2000). Sialic acid when present in the LPS of NTHi strains, as a terminal residue on selected oligosaccharide extensions, has been shown to contribute significantly to bacterial resistance to the killing effect of normal human serum (Hood et al., 1999, 2001b; Månsson et al., 2001). The consistent and significant reduction in the resistance to killing by normal human serum observed with mutation of lgtF in a number of strain backgrounds in our serum resistance experiments was unexpected. It has recently been shown that previously uncharacterized tetrasaccharide units, including a sialic acid residue, can be found as an extension from the glucose attached to HepI in a minority of glycoforms in strains RM118 and RM153 under appropriate growth conditions (Cox et al., 2002) (Table 1). These cryptic glycoforms could explain changes seen in serum resistance for strains mutated in lgtF, but would not explain the results seen with strain 375, a strain that shows no structural or genetic evidence of these atypical glycoforms.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 November 2003; accepted 14 December 2003.



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