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.     

Haemophilus influenzae type b strain A2 has multiple sialyltransferases involved in lipooligosaccharide sialylation

The lipooligosaccharide (LOS) of Haemophilus influenzae contains sialylated glycoforms, and a sialyltransferase, Lic3A, has been previously identified. We report evidence for two additional sialyltransferases, SiaA, and LsgB, that affect N-acetyllactosamine containing glycoforms. Mutations in genes we have designated siaA and lsgB affected only the sialylated glycoforms containing N-acetylhexosamine. A mutation in siaA resulted in the loss of glycoforms terminating in sialyl-N-acetylhexosamine and the appearance of higher molecular weight glycoforms, containing the addition of phosphoethanolamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Chromosomal complementation of the siaA mutant resulted in the expression of the original sialylated LOS phenotype. A mutation in lic3A resulted in the loss of sialylation only in glycoforms lacking N-acetylhexosamine and had no effect on sialylation of the terminal N-acetyllactosamine epitope. A double mutant in siaA and lic3A resulted in the complete loss of sialylation of the terminal N-acetyllactosamine epitope and expression of the higher molecular weight sialylated glycoforms seen in the siaA mutant. Mutation of lsgB resulted in persistence of sialylated glycoforms but a reduction in N-acetyllactosamine containing glycoforms. A triple mutant of siaA, lic3A, and lsgB contained no sialylated glycoforms. These results demonstrate that the sialylation of the LOS of H. influenzae is a complex process involving multiple sialyltransferases.     

Ultrastructural characterization of capsulated Haemophilus influenzae type b and two spontaneous nontypable mutants.

Capsulated Haemophilus influenzae type b and two spontaneous mutants (classes I and II variants) were characterized by transmission and scanning electron microscopy. When cells were treated with type b-specific antiserum prior to manipulations for electron microscopy, sectioned capsulated cells had electron-dense, fibrous capsular antigen-antibody complexes around them. In negatively stained preparations, the complexes appeared as electron-transparent zones surrounding cells. In contrast, only residual electron-dense, extracellular material was seen in sectioned, untreated, capsulated cells, and electron-dense "bridges" connected adjacent cells in negatively stained preparations. No extracellular capsular material was seen around the class I and II variants. Characteristic electron-translucent regions were always observed within the cytosol of the class I cells, both in thin sections and by negative staining. These areas were located adjacent to the cell envelope separating the plasma membrane from the dense cytoplasmic matrix. At times, electron-dense, thread-like material extended from the dense cytoplasmic matrix to the plasma membrane. No such regions were seen in the capsulated and class II cells. Class I cells fixed with methanol or suspended in NaCl or phosphate-buffered saline prior to treatment with fluorescein-tagged type b-specific antiserum (FTA reagent) exhibited, by immunofluorescence, patches of capsular antigen along their sides. However, when fixed with glutaraldehyde or OsO4 or suspended in tris-(hydroxymethyl)aminomethane plus Ca2+ buffer prior to treatment with FTA reagent, no patches of capsular antigen were seen. Subsequent exposure of the latter cells to methanol followed by treatment with FTA reagent resulted in the reappearance of the patches of capsular antigen. Thus, in the class I variant the capsular antigen is unlikely to be surface located. Scanning electron microscopy revealed that class I and II variant cells within undisturbed colonies were regularly aligned side-by-side, whereas cells within colonies of the capsulated strain were randomly distributed.     

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.     

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.     

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.     

Human serum bactericidal activity against Haemophilus influenzae type b

We examined bactericidal and opsonizing activity of pooled adult 'immune' serum against Haemophilus influenzae type b with and without the addition of phagocytes. Four type b strains from cerebrospinal fluid (CSF) and three such strains from the nasopharynx (NP) of healthy children were examined. Duplicate reaction mixtures contained organisms in exponential (E) or stationary phase (S) of growth, serum, a complement source (human agammaglobulinaemic serum), and culture medium (bactericidal assay); separate assays contained the above components and polymorphonuclear leucocytes (opsonization system). A decrease in bacterial density of greater than or equal to 1 log10 unit was considered significant. All four S-CSF strains, three of four E-CSF strains and one of three S-NP strains were sensitive to the bactericidal activity of pooled serum. The other E-CSF strain, two S-NP strains and all three E-NP strains were resistant to the bactericidal activity of pooled serum. Two of three E-NP strains were opsonized by pooled serum; the other strains resistant to the bactericidal activity of pooled serum were also resistant to opsonization. Bactericidal and opsonizing activity of serum from an immunized adult was greater than or equal to that of pooled serum against each strain. Assuming normal adults are immune to invasive H. influenzae type b infection, an experimental test reflecting this immunity is the bactericidal activity against CSF isolates tested in stationary phase. We conclude that protection against invasive disease due to H. influenzae type b appears more complex than the presence of bactericidal and opsonizing activity in serum.     

Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b

The specificity by which Haemophilus species acquired iron from transferrin (TF) was investigated. In a plate bioassay H. influenzae used iron bound to human, bovine and rabbit TFs but not mouse, rat, dog, horse, guinea-pig, pig or ovo- TFs or human and bovine lactoferrins. In contrast, H. pleuropneumoniae used iron only from pig TF whilst H. parainfluenzae was unable to utilize iron bound to any of the human or animal TFs tested. The inhibition of growth imposed on H. influenzae type b strain Eagan by the addition of the synthetic iron chelator EDDA to the culture medium was reversed by 30% iron-saturated human TF added directly to the medium but not when the TF was contained inside a dialysis bag. Dot-blotting of whole cells revealed that human TF bound to the surface of bacteria cultured in iron-restricted but not in iron-plentiful media. Incubation of whole bacterial cells in the presence of the proteolytic enzyme trypsin also abolished TF-binding activity, suggesting that the TF receptor was a protein. In competition dot blotting experiments, human and bovine but not rabbit, dog, mouse or guinea-pig TFs blocked the binding of a horseradish peroxidase--human TF conjugate. SDS-PAGE and Western blotting of outer membranes revealed the presence of a TF-binding protein of approximately 72 kDa. These results suggest that the acquisition of TF-bound iron by H. influenzae type b probably involves a direct interaction with an outer-membrane protein which shows some TF-species specificity.