Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.     

Tn916 insertion mutagenesis in Escherichia coli and Haemophilus influenzae type b following conjugative transfer.

Transposon Tn916 was shown to be capable of direct conjugative transfer in broth and membrane matings between strains of Escherichia coli K12 and between E. coli K12 and Haemophilus influenzae type b. Only Tn916 was transferred, but Tn916 donor ability was not itself inheritable by the recipients and seemed to be associated with the presence of Tn916 on a non-conjugative pBR322-derived vector in the original donor strain. Transfer of Tn916 by conjugation was found to be an efficient method for producing insertion mutations in the chromosome of recipient cells. Although such insertions were unstable when the cells were grown under non-selective conditions, it was possible to show that over 40% of the isolated Tn916 insertions in the chromosome of E. coli K12 were in gene(s) concerned with histidine biosynthesis, implying that there is a partial hot-spot for Tn916 insertion on the E. coli K12 chromosome. When a strain of H. influenzae type b was used as a recipient, out of approximately 1500 transconjugants tested, two mutants were isolated with insertions in genes controlling the expression of iron-regulated transferrin-binding proteins. These mutants constitutively produced major 76 kDa and minor 90 kDa proteins which bound transferrin, even when grown under iron-sufficient conditions. Tn916 insertion mutagenesis, following transfer by conjugation, is a convenient method for isolating mutations in genes concerned with iron acquisition by this important human pathogen.     

Generation of lipooligosaccharide mutants of Haemophilus influenzae type b.

We previously reported the analysis of recombinant plasmids from Haemophilus influenzae type b (Hib) that lead to modifications of Escherichia coli lipopolysaccharide (LPS) (Y. Abu Kwaik, R. E. McLaughlin, M. A. Apicella, and S. M. Spinola, Mol. Microbiol. 5:2475-2480, 1991). The modified LPS species are recognized by monoclonal antibodies (MAbs) 6E4 and 3F11. MAb 6E4 binds to a stable 2-keto-3-deoxyoctulosonic acid epitope, while MAb 3F11 binds to a Gal beta 1-4GlcNac epitope that phase varies in Hib at a frequency of 2 to 5%. The internal EcoRI fragment containing most of the DNA required for LPS modification in E. coli was used as the target for transposon mutagenesis. Plasmids containing minitransposon m-Tn3(Cm) randomly inserted into the target fragment were transformed into the isogenic Hib strain, and transposon integration into the Hib chromosome was verified by colony hybridization. The lipooligosaccharides of 36 transformants were phenotypically and antigenically characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with a variety of MAbs that recognize both stable and phase-varying lipooligosaccharide epitopes. The majority of the mutants had altered reactivity with MAb 6E4. With one exception, these mutants retained the ability to express phase-varying epitopes. Analysis of the transformants suggested that the 6E4 epitope was contained on an oligosaccharide chain separate from that of phase-varying epitopes and appeared to be assembled in at least three separate steps.     

Transport of hemin byHaemophilus influenzae type b

Haemophilus influenzae may be distinguished from other gram-negative bacteria by its growth requirement for hemin. The ability of this bacterium to accumulate hemin while growing in a fully defined medium has been partially characterized.Haemophilus influenzae type b ATCC 9795 transported hemin at a rate of 1.2 pmol/min/10⁹ cells during logarithmic growth. The kinetics of active transport of doubly radiolabeled hemin indicated that both iron and the porphyrin ring were taken up at the same rate. Hemin satisfied some of the total iron requirement ofHaemophilus as determined by starving the cells for iron with the addition of ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA) and by limiting the porphyrin supply. Outer membrane proteins were compared from cells grown under hemin sufficiency versus cells grown under hemin starvation: in the latter case, a protein of molecular weight 43,000 was present in enhanced amounts; this protein may play a role in the permeability of hemin across the cell envelope ofH. influenzae type b.     

Epidemiology of Haemophilus influenzae type b invasive disease in Wales.

OBJECTIVE--To investigate the epidemiology of invasive disease due to Haemophilus influenzae type b, the clones responsible, and the antibiotic resistance of the isolates. DESIGN--Prospective population based analysis of clinical and epidemiological data collected for Gwynedd during 1980-90 and in the whole of Wales during 1988-90. SETTING--19 hospitals in Wales; all medical microbiology laboratories in Wales participated. PATIENTS--82 patients with confirmed invasive infections caused by H influenzae type b in Gwynedd during 1980-90 and 207 in Wales during 1988-90. MAIN OUTCOME MEASURES--Clinical and epidemiological measures; analysis of the clonal types of the isolates based on the electrophoretic mobilities of 17 metabolic enzymes; and antibiotic resistance. RESULTS--The annual incidence of H influenzae type b infections in Gwynedd was 3.2 cases/100,000 and in Wales was 2.5 cases/100,000. Most cases occurred in children aged under 5 years, the highest annual incidence being in those aged under 1 (84.6/100,000 and 56.9/100,000 in Wales). The cumulative risk of acquiring H influenzae type b disease by the fifth birthday was one in 456 in Gwynedd and one in 578 in Wales. Fifteen per cent of cases in Gwynedd and 7% of those in Wales occurred in adults. Predominant clinical conditions were meningitis in children and pneumonia in adults. In Gwynedd 2/70 (3%) children and 5/12 (42%) adults died. Long term neurological sequelae occurred in 8% (4/48) of children who survived haemophilus meningitis. Children presenting with infection were usually the youngest members of their family. No secondary household cases were identified. 100 of 128 (78%) strains were of a single clone, electrophoretic type 12.5, and 4/207 (1.9%) isolates from Wales were resistant to both ampicillin and chloramphenicol. CONCLUSIONS--The annual rate of infection in children aged under 5 in four Welsh counties was 12-44% higher than that previously published for the United Kingdom. The study emphasises the potential value of a vaccine effective in early infancy and provides baseline data to assess its efficacy after its introduction. Alternatives to ampicillin and chloramphenicol should be used as first line, empirical treatment for severe infections that might be caused by H influenzae type b in Wales.     

Haemophilus influenzae type b polysaccharide-protein conjugate vaccine

An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.     

Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection.

Outer-membrane proteins (OMPs) from Haemophilus influenzae type b (strain Eagan), grown both in vitro (broth) and in vivo (rat intra-peritoneal), were separated by SDS-PAGE. The major OMPs were present in both growth conditions although the amounts of OMP a and OMP d were reduced in rat-grown organisms. There were strong additional bands in in-vivo-grown organisms at 51 and 92 kDa. Antiserum was raised in rabbits against in-vivo-grown bacteria, and absorbed with lysates of in-vitro-grown bacteria. This serum was used in Western blot analysis of OMPs from in-vitro- and in-vivo-grown cells to identify immunogenic proteins present in infection. These infection-associated OMPs had apparent molecular masses of 43 kDa, 48 kDa, 81 kDa and greater than 200 kDa. Bands of reactivity, of the same molecular mass as some of these, were found on immunoblots when rat and human convalescent sera were used as the source of primary antibody. In particular, a band of 81 kDa was recognized by pooled rat and three human convalescent sera.     

A physical map of the genome of Haemophilus influenzae type b.

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (PFGE) was used in combination with Southern hybridization to construct a genomic restriction map for the pathogen Haemophilus influenzae serotype b, strain Eagan. Sites for four restriction endonucleases, EagI, NaeI, RsrII and SmaI, were located on the 2100 kbp circular chromosome. Twelve potential virulence loci have been placed on the map together with certain loci essential for growth of the bacteria (e.g. ribosomal RNA operons). PFGE also provided a valuable tool for characterizing ten capsulated, type b isolates (other than Eagan) known to be genetically heterogeneous and two laboratory-derived variants (transformants) derived through complex recombinational events involving random uptake of high-molecular-mass donor genomic DNA.     

Immunogenicity of outer membrane derivatives ofHaemophilus influenzae type b

We prepared outer membrane derivatives ofHaemophilus influenzae type b to determine whether the residual capsular and noncapsular surface components are immunogenic and protective. These fragments consist primarily of six major proteins and lipopolysaccharide. By transmission electron microscopy, they appeared as small, membrane-like fragments or larger, cellshaped double-track ghosts. Rabbits immunized with ghosts responded with increases in serum anticapsular antibody and bactericidal activity. Antisera absorbed with capsular antigen to remove anticapsular antibody remained bactericidal and passively protected infant rats. These data suggest that antibodies to noncapsular surface antigens are protective, and that outer membrane derivatives retain some of the constituents responsible for stimulating immunity.     

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.