The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

Griffin, Ruth; Cox, Andrew D.; Makepeace, Katherine; Richards, James C.; Moxon, E. Richard; Hood, Derek W.

doi:10.1099/mic.0.26387-0

Cited by 28 publications

(39 citation statements)

References 29 publications

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“…The phase-variable lic2A and licA genes, encoding a galactosyltranferase and choline kinase, respectively, are present in the strain 86-028NP genome (45,48,117). The lex2B gene, which encodes a glucosyltransferase in the serotype b strain DL42 as well as a number of other serotypeable strains, is present in the strain 86-028NP genome (41,53). Five-prime to the lex2B gene in strain DL42 is the short phase-variable lex2A gene.…”

Section: Resultsmentioning

confidence: 99%

Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae : Comparative Study with H. influenzae Serotype d, Strain KW20

Harrison¹,

et al. 2005

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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.In 1995 Haemophilus influenzae strain Rd, a rough derivative of H. influenzae serotype d strain KW20 (strain Rd hereafter), became the first free-living organism to have its genome sequenced to completion (34). Importantly, this also helped establish the large-scale shotgun approach, mated with the utilization of a scaffolding library and computer-assisted assembly, as a rational and expeditious approach for the sequencing of small bacterial genomes. Strain Rd was chosen as the prototypic bacterium for complete genome sequencing as it has a genome size representative of other bacteria and a GϩC content close to that of the human genome. Additionally, at the time of sequencing, a physical map of the strain Rd genome did not exist, so this genome was a good test for the approach of shotgun sequencing, scaffolding, and assembly (34).Although strain Rd is the exemplar organism for the current small-genome sequencing rationale and an important model organism for studying H. influenzae biology, strain Rd is a poor model for the study of pathogenicity caused by members of the genus Haemophilus. Serotype b strains of H. influenzae cause invasive diseases, for example, meningitis, and nontypeable H. influenzae (NTHi) strains principally have a role in localized respiratory disease, particularly in otitis media, acute sinusitis, and community-acquired pneumonia and have important conseque...

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Section: Resultsmentioning

confidence: 99%

Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae : Comparative Study with H. influenzae Serotype d, Strain KW20

Harrison¹,

et al. 2005

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“…1) (4, 5). A type b clinical strain, RM153, used in previous virulence studies (9, 16), typically generates LPS molecules containing only four hexose sugars (17), depending on whether lic2A, lgtC, or lex2 is out of frame (4,7,10). This is in contrast to the related strain RM7004, which has up to nine hexose sugars in its LPS due to these three loci being predominantly in frame (Fig.…”

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confidence: 99%

Digalactoside Expression in the Lipopolysaccharide of Haemophilus influenzae and Its Role in Intravascular Survival

et al. 2005

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Digalactoside (gal␣-1-4 gal␤) structures of the lipopolysaccharide (LPS) of Haemophilus influenzae are implicated in virulence. A confounding factor is that tetranucleotide repeats within the lic2A, lgtC, and lex2 genes mediate phase-variable expression of the digalactosides. By deleting these repeats, we constructed recombinant strains of RM153 constitutively expressing either one or two LPS digalactosides. Expression of two digalactosides, rather than one, was associated with increased virulence of H. influenzae in vivo.Lipopolysaccharide (LPS) is one of the major virulence determinants of the human pathogen Haemophilus influenzae. The number of hexoses and phosphate groups replacing the triheptose (HepI to HepIII) backbone ( Fig. 1) is variable within any strain owing to high-frequency translational switching (phase variation) of LPS genes containing repeat tracts. Phase-variable LPS genes include lic2A and lgtC, which are involved in the assembly of gal␣-1-4 gal␤ into the oligosaccharide extensions from the conserved triheptose backbone (7, 10), and lex2, which is involved in completion of the HepIattached diglucoside acceptor for this digalactoside ( Fig. 1) (4). The investigation of the association between digalactoside expression on H. influenzae LPS and virulence has relied on the monoclonal antibody (MAb) 4C4 for detecting the expression of the digalactoside (1, 2, 12, 13). The findings from these studies have been difficult to interpret because of the confounding factor of phase variation and the different numbers and locations of the digalactoside on oligosaccharide extensions in different strains (7,17,18).Recently, it has been shown that MAb 4C4 binds digalactosides that are part of the extensions from both HepI and HepII of the triheptose LPS backbone (Fig. 1) (4, 5). A type b clinical strain, RM153, used in previous virulence studies (9, 16), typically generates LPS molecules containing only four hexose sugars (17), depending on whether lic2A, lgtC, or lex2 is out of frame (4,7,10). This is in contrast to the related strain RM7004, which has up to nine hexose sugars in its LPS due to these three loci being predominantly in frame (Fig. 1). Knowing the LPS structure, the genes required for digalactoside assembly, and the fact that tetranucleotide repeats mediate phase variation, we constructed recombinant strains of RM153 in which variable expression of digalactosides was eliminated by removing the repeat tracts located within each of these genes. Briefly, two isogenic strains were constructed by transformation (6) using appropriate chromosomal DNA or plasmid constructs in which the repeats had been deleted for lic2A (8), lex2 (5), or lgtC. All three genes were constitutively expressed in the first strain, RM153lic2AϩtlgtCϩlex2ϩk, to facilitate expression of two digalactosides, while in the second strain, RM153lic2AϩtlgtCϩlex2Ϫk, lex2 was mutated such that only a single digalactoside in the extension from HepII was expressed (Fig.

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“…A potential candidate, CJJ1165 (Table 1), was located between waaV and gmhA1 (19). This ORF encoded a predicted 252-amino-acid protein with a molecular mass of 61 kDa that was 35% identical to a ␤-1,4-glucosyltransferase, Lex2B, from H. ducreyi (11). This 81-176 ORF was disrupted by insertional mutagenesis of a clone of this region, as described in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

Genetic Analysis of Lipooligosaccharide Core Biosynthesis in Campylobacter jejuni 81-176

et al. 2008

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We report isolation and characterization of Campylobacter jejuni 81-176 lgtF and galT lipooligosaccharide (LOS) core mutants. It has been suggested that the lgtF gene of C. jejuni encodes a two-domain glucosyltransferase that is responsible for the transfer of a ␤-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a ␤-1,2-glucose residue on Hep II. A site-specific mutation in the lgtF gene of C. jejuni 81-176 resulted in expression of a truncated LOS, and complementation of the mutant in trans restored the core mobility to that of the wild type. Mass spectrometry and nuclear magnetic resonance of the truncated LOS confirmed the loss of two glucose residues, a ␤-1,4-glucose on Hep I and a ␤-1,2-glucose on Hep II. Mutation of another gene, galT, encoding a glycosyltransferase, which maps outside the region defined as the LOS biosynthetic locus in C. jejuni 81-176, resulted in loss of the ␤-(1,4)-galactose residue and all distal residues in the core. Both mutants invaded intestinal epithelial cells in vitro at levels comparable to the wild-type levels, in marked contrast to a deeper inner core waaC mutant. These studies have important implications for the role of LOS in the pathogenesis of Campylobacter-mediated infection.

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The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

Cited by 28 publications

References 29 publications

Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae : Comparative Study with H. influenzae Serotype d, Strain KW20

Genomic Sequence of an Otitis Media Isolate of Nontypeable Haemophilus influenzae : Comparative Study with H. influenzae Serotype d, Strain KW20

Digalactoside Expression in the Lipopolysaccharide of Haemophilus influenzae and Its Role in Intravascular Survival

Genetic Analysis of Lipooligosaccharide Core Biosynthesis in Campylobacter jejuni 81-176

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