Haemophilus influenzae: capsule vaccine and capsulation genetics

In 1931, Dr Margaret Pittman reported her discovery that Haemophilus influenzae strains responsible for meningitis had a polysaccharide capsule, and that one capsular type, serotype b, was responsible for nearly all cases. Diverse programmes of research aimed at understanding and exploiting this seminal observation culminated, in the 1980s, in the introduction of a purified type b polysaccharide vaccine to protect children against this terrible disease. Subsequent improvements in vaccine immunogenicity have translated into impressive efficacy and the suggestion that, were all children to be immunized, a major cause of life-threatening childhood infection might be vanquished.     

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.     

Capsulation in distantly related strains of Haemophilus influenzae type b: genetic drift and gene transfer at the capsulation locus.

Among natural populations of capsulate Haemophilus influenzae, clones of strains with type b capsular polysaccharide are found in each of two widely separated phylogenetic divisions. The chromosomal capsulation locus found in strains from either division has a three-segment organization, with serotype-specific DNA nested between elements common to all serotypes, but pairwise comparison of the segments between the divisions suggests that they have distinct phylogenetic histories. Genes clustered in one of the non-serotype-specific segments appear to have diverged from an ancestral element, reflected in 12% nucleotide sequence divergence in one homologous pair. In contrast, genes conferring the capacity to produce type-specific polysaccharide exhibit no such divergence, and we speculate that these have been subject more recently to horizontal transfer within the bacterial population. Clinically important capsulate gram-negative bacteria share a common organization of their capsulation loci, arguing convergence on a successful arrangement of genes. In H. influenzae this appears to have allowed the occasional exchange of serotype-specific capsulation genes between strains, a event of potential clinical importance in this major bacterial pathogen.     

The identification a novel gene required for lipopolysaccharide biosynthesis by Haemophilus influenzae RM7004, using transposon Tn916 mutagenesis

Abstract Mutagenesis with the transposon Tn916 was used as a strategy to identify genes required for synthesis of the Galα(1–4)βGal component of Haemophilus influenzae strain RM7004 lipopolysaccharide. Insertion of Tn916 into an open reading frame (ORF) encoding a protein with 75% homology to the Escherichia coli methionine related protein (Mrp) is described. Mutations in mrp resulted in loss of reactivity with monoclonal antibody (mAb) 4C4, which recognises Galα(1–4)βGal, and expression of LPS with a different electrophoretic profile to that of wild-type RM7004. An unexpected feature of this mutation was that it appeared to influence the number of copies of 5'-CAAT-3' present in lic2A , a gene which is also required for biosynthesis and phase variable expression of the Galα(1–4)βGal LPS epitope.     

Region II of the Haemophilus influenzae Type b capsulation locus as involved in serotype-specific polysaccharide synthesis

The central (serotype-specific) Region II of the Haemophilus influenzae Type b capsulation locus cap is 8.3 kb long and contains a cluster of four genes. We show that these genes, designated orf1 to orf4, are involved in the biosynthetic steps required for the formation of the Type b capsular polysaccharide and that orf1 probably encodes a CDP-ribitolpyrophosphorylase. We present evidence that growth of polysaccharide chains takes place through the alternating addition of single sugar nucleotides.     

Plasmid containing a DNA ligase gene from Haemophilus influenzae

A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2 . Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant.     

acs1 of Haemophilus influenzae type a capsulation locus region II encodes a bifunctional ribulose 5-phosphate reductase- CDP-ribitol pyrophosphorylase

The serotype-specific, 5.9-kb region II of the Haemophilus influenzae type a capsulation locus was sequenced and found to contain four open reading frames termed acs1 to acs4. Acs1 was 96% identical to H. influenzae type b Orf1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, Mol. Microbiol. 15:107-118, 1995). Low but significant homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme. To test this hypothesis, acs1 was cloned in an expression vector and overexpressed in Escherichia coli. Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns. The dehydrogenase activity was specific for ribulose 5-phosphate and NADPH in one direction and for ribitol 5-phosphate and NADP+ in the other direction and was markedly stimulated by CTP. The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate. We conclude that acs1 encodes a bifunctional enzyme that converts ribulose 5-phosphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the H. influenzae type a capsular polysaccharide.     

Evidence for gene silencing in Haemophilus influenzae

Evidence for gene silencing of Haemophilus influenzae involved a beta-subunit of RNA polymerase. The gene presumed silenced was rifampin resistance. The evidence that it was silencing, rather than dominance of a rifampin-sensitive marker, was that it took place when the rifampin resistance marker was on both a plasmid and the chromosome, without the presence of a rifampin-sensitive marker, as judged by lack of transformation of a rifampin-resistant cell to rifampin sensitivity by the plasmid. In addition, three compounds that are known to decrease gene silencing in eukaryotes (trichostatin A, sodium butyrate and 5-azacytidine) also decreased the presumed silencing in H. influenzae. Silencing of rifampin-resistant Escherichia coli did not take place with the plasmid from H. influenzae.     

The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl-N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.     

A gene cluster involved in the utilization of both free heme and heme:hemopexin by Haemophilus influenzae type b.

The utilization of heme bound to the serum glycoprotein hemopexin by Haemophilus influenzae type b (Hib) strain DL42 requires the presence of the 100-kDa heme:hemopexin-binding protein encoded by the hxuA gene (M. S. Hanson, S. E. Pelzel, J. Latimer, U. Muller-Eberhard, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 89:1973-1977, 1992). Nucleotide sequence analysis of a 5-kb region immediately upstream from the hxuA gene revealed the presence of two genes, designated hxuC and hxuB, which encoded outer membrane proteins. The 78-kDa HxuC protein had similarity to TonB-dependent outer membrane proteins of other organisms, whereas the 60-kDa HxuB molecule most closely resembled the ShlB protein of Serratia marcescens. A set of three isogenic Hib mutants with cat cartridges inserted individually into their hxuA, hxuB, and hxuC genes was constructed. None of these mutants could utilize heme:hemopexin. The hxuC mutant was also unable to utilize low levels of free heme, whereas both the hxuA and hxuB mutants could utilize free heme. When the wild-type hxuC gene was present in trans, the hxuC mutant regained its ability to utilize low levels of free heme but still could not utilize heme:hemopexin. The hxuA mutant could utilize heme:hemopexin when a functional hxuA gene from a nontypeable H. influenzae strain was present in trans. Complementation analysis using this cloned nontypeable H. influenzae hxuA gene also indicated that the HxuB protein likely functions in the release of soluble HxuA from the Hib cell. These studies indicate that at least two and possible three gene products are required for utilization of heme bound to hemopexin by Hib strain DL42.     

Radiation Sensitivity of Haemophilus influenzae: a Composite Response

The survival of ultraviolet (UV)-irradiated cultures of Haemophilus influenzae Rd is determined by at least two responses: (i) excision-repair ability and (ii) UV-induced cell lysis. An UV-resistant mutant, BC200, has the same capabilities as the wild type, Rd, for excising dimers but does not exhibit lysis. Lytic response is dose-dependent. Relative to the wild type, a lower dose of UV causes lysis of a UV-sensitive mutant, BC100, which is incapable of excising thymine dimers. A lytic protein is present in cultures undergoing lysis. Synthesis of this protein is initiated 45 to 60 min after irradiation. Lysis appears to be due to derepression of a defective prophage which codes for an endolysin-like lytic enzyme.