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

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.     

The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5'-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17-38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.     

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.     

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.     

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

Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and 1H NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1--> 3)-L-alpha-D-He pp-(1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI.     

Destabilization of tetranucleotide repeats in Haemophilus influenzae mutants lacking RnaseHI or the Klenow domain of PolI

A feature of Haemophilus influenzae genomes is the presence of several loci containing tracts of six or more identical tetranucleotide repeat units. These repeat tracts are unstable and mediate high frequency, reversible alterations in the expression of surface antigens. This process, termed phase variation (PV), enables H.influenzae to rapidly adapt to fluctuations in the host environment. Perturbation of lagging strand DNA synthesis is known to destabilize simple sequence repeats in yeast and Escherichia coli. By using a chromosomally located reporter construct, we demonstrated that the mutation of an H.influenzae rnhA (encoding RnaseHI) homologue increases the mutation rates of tetranucleotide repeats ~3-fold. Additionally, deletion of the Klenow domain of DNA polymerase I (PolI) resulted in a ~35-fold increase in tetranucleotide repeat-mediated PV rates. Deletion of the PolI 5′>3′ exonuclease domain appears to be lethal. The phenotypes of these mutants suggest that delayed or mutagenic Okazaki fragment processing destabilizes H.influenzae tetranucleotide repeat tracts.     

Tetrameric repeat units associated with virulence factor phase variation in Haemophilus also occur in Netsseria spp. and Moraxella catarrhalis

Abstract The tetrameric repeat units 5'-CAAT-3' and 5'-GCAA-3' are associated with phase variable expression of lipopolysaccharide biosynthetic genes in Haemophilus influenzae . Four other tetrameric repeat units have also been reported from H. influenzae strain Rd, 5'-CAAC-3', 5'-GACA-3', 5'-AGCT-3', and 5'-TTTA-3', which are also associated with putative virulence factors. Using oligonucleotide probes corresponding to five tandem copies of each of these tetramers, we have screened three strains of Neisseria meningitidis and one each of Neisseria gonorrhoeae, Neisseria lactamica, Haemophilus parainfluenzae, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiceptica and Moraxella catarrhalis for the presence of these motifs. We have demonstrated the presence of multiple copies of the 5'-GCAA-3' motif in all the Neisseria strains tested, and also the repeated motif 5'-CAAC-3' in M. catarrhalis . We have further demonstrated by Southern blot analysis that the 5'-CAAC-3' repeats detected in M. catarrhalis are probably associated with the same genes as in H. influenzae , but that the 5'-GCAA-3' motifs in N. meningitidis are not. The use of characterised tetrameric DNA sequences as hybridisation probes may prove useful in the identification of novel phase variable virulence determinants in organisms other than H. influenzae .     

The molecular mechanism of phase variation of H. influenzae lipopolysaccharide

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

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.     

An alternate pattern for globoside oligosaccharide expression in Haemophilus influenzae lipopolysaccharide: structural diversity in nontypeable strain 1124

Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are 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) of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEA-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEA-->4]-alpha-Kdo-(2-->6)-lipid A. We report here structural studies of LPS from nontypeable H. influenzae strain 1124 expressing these motifs linked to both the proximal heptose (HepI) and HepIII at the same time. This novel finding was obtained by structural studies of LPS using NMR techniques and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MS(n)() on permethylated dephosphorylated OS. The use of defined mutants allowed us to confirm structures unambiguously and understand better the biosynthesis of each of the globotetraose units. We found that lgtC is involved in the expression of alpha-d-Galp-(1-->4)-beta-d-Galp in both extensions, whereas lic2A directs only the expression of beta-d-Galp-(1-->4)-beta-d-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MS/MS. A common sialylated structure in H. influenzae LPS is sialyllactose linked to HepIII. This structure exists in strain 1124. However, results for the lpsA mutant indicate that sialyllactose extends from HepI as well, a molecular environment for sialyllactose in H. influenzae that has not been reported previously. In addition, the LPS was found to carry phosphorylcholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII.     

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod–res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.     

Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.     

Infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable Haemophilus influenzae

Infectious exacerbations of chronic obstructive pulmonary disease (COPD) have been reported to occur with both viral and bacterial pathogens. In this study, 35 exacerbations associated with the isolation of non-typeable Haemophilus influenzae from sputum were identified as part of a prospective longitudinal study. Samples from these patients were subjected to immunoassays to identify a new immune response to the homologous isolate of non-typeable H. influenzae to more accurately assess a bacterial etiology. These patients also were studied carefully for evidence of viral infection using viral culture, serology and polymerase chain reaction-based assays. Sixteen of 35 exacerbations (45.7%) were associated with evidence of acute viral infection and 11 of the 35 exacerbations (31.4%) were associated with the development of new serum IgG to homologous non-typeable H. influenzae. Overall, evidence of infection with a respiratory virus or non-typeable H. influenzae was seen in 24 of 35 exacerbations (68.6%). No association between viral infection and immune response to non-typeable H. influenzae was observed, although a trend toward an immune response to non-typeable H. influenzae and absence of viral infection was seen. The results show that exacerbations in adults with COPD were associated with infection caused by virus alone, non-typeable H. influenzae alone, or virus and non-typeable H. influenzae simultaneously.     

Genes of non-typeable Haemophilus influenzae expressed during interaction with human epithelial cell lines

Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.     

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.     

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

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

Mutations in polI but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats

Haemophilus influenzae (Hi), an obligate upper respiratory tract commensal/pathogen, uses phase variation (PV) to adapt to host environment changes. Switching occurs by slippage of nucleotide repeats (microsatellites) within genes coding for virulence molecules. Most such microsatellites in Hi are tetranucleotide repeats, but an exception is the dinucleotide repeats in the pilin locus. To investigate the effects on PV rates of mutations in genes for mismatch repair (MMR), insertion/deletion mutations of mutS, mutL, mutH, dam, polI, uvrD, mfd and recA were constructed in Hi strain Rd. Only inactivation of polI destabilized tetranucleotide (5'AGTC) repeat tracts of chromosomally located reporter constructs, whereas inactivation of mutS, but not polI, destabilized dinucleotide (5'AT) repeats. Deletions of repeats were predominant in polI mutants, which we propose are due to end-joining occurring without DNA polymerization during polI-deficient Okazaki fragment processing. The high prevalence of tetranucleotides mediating PV is an exceptional feature of the Hi genome. The refractoriness to MMR of hypermutation in Hi tetranucleotides facilitates adaptive switching without the deleterious increase in global mutation rates that accompanies a mutator genotype.     

lpt6, a gene required for addition of phosphoethanolamine to inner-core lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae

We previously described a gene, lpt3, required for the addition of phosphoethanolamine (PEtn) at the 3 position on the beta-chain heptose (HepII) of the inner-core Neisseria meningitidis lipopolysaccharide (LPS), but it has long been recognized that the inner-core LPS of some strains possesses PEtn at the 6 position (PEtn-6) on HepII. We have now identified a gene, lpt6 (NMA0408), that is required for the addition of PEtn-6 on HepII. The lpt6 gene is located in a region previously identified as Lgt-3 and is associated with other LPS biosynthetic genes. We screened 113 strains, representing all serogroups and including disease and carriage strains, for the lpt3 and lpt6 genes and showed that 36% contained both genes, while 50% possessed lpt3 only and 12% possessed lpt6 only. The translated amino acid sequence of lpt6 has a homologue (72.5% similarity) in a product of the Haemophilus influenzae Rd genome sequence. Previous structural studies have shown that all H. influenzae strains investigated have PEtn-6 on HepII. Consistent with this, we found that, among 70 strains representing all capsular serotypes and nonencapsulated H. influenzae strains, the lpt6 homologue was invariably present. Structural analysis of LPS from H. influenzae and N. meningitidis strains where lpt6 had been insertionally inactivated revealed that PEtn-6 on HepII could not be detected. The translated amino acid sequences from the N. meningitidis and H. influenzae lpt6 genes have conserved residues across their lengths and are part of a family of proven or putative PEtn transferases present in a wide range of gram-negative bacteria.