Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions.

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.     

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.     

Outer membrane protein P6 of Haemophilus influenzae binds to its own gene

Outer membrane protein P6 is an important antigen expressed on the surface of all strains of Haemophilus influenzae. The predicted amino acid sequence of P6 contains a region of alpha helices that shares sequence identity with a family of helix-turn-helix DNA-binding proteins. A search for sequence-specific binding sites that resemble an operator region within the gene revealed a sequence with striking homology to the consensus operator sequence for lambda Cro and repressor. To test the hypothesis that P6 binds its own gene, purified P6 on nitrocellulose was probed with plasmid DNA containing the P6 gene. P6 bound the P6 gene in this Southwestern blot assay. To further test the observation, gel shift analysis was performed. Gel shift assays using a P6-specific monoclonal antibody demonstrated that P6 in crude cell extracts binds to the region of the gene containing the putative binding site. Competition with a synthetic oligonucleotide corresponding to the putative binding site inhibited binding of P6 to the P6 gene on nitrocellulose and in the gel shift assay. In addition, this oligonucleotide bound directly to P6 on nitrocellulose. Finally, DNase footprinting confirmed that P6 bound specifically to the same region of the P6 gene. These results indicate that P6 binds to a sequence-specific site within its own gene, suggesting that P6 regulates its own expression. This represents the first example of a Gram-negative outer membrane protein binding to its own gene and has potentially important implications as a mechanism for regulation of expression of outer membrane antigens.     

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 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.     

Diversity of the outer membrane protein P2 gene from major clones of Haemophilus influenzae type b

In previous studies, it has been demonstrated that outer membrane protein P2 from Haemophilus influenzae type b has porin activity and that antibody directed against P2 is protective in an infant rat bacteraemic model. Outer membrane protein subtyping has been employed to subclassify type b Haemophilus isolates. Strain MinnA has the outer membrane protein subtype 1H and is representative of the dominant clonal group of disease-producing isolates in the United States. In the present study, the P2 gene from strain MinnA was employed to probe EcoRI- and Pvull-digested chromosomal DNA from 24 Haemophilus influenzae type b isolates representative of the common outer membrane protein subtype groups observed throughout the world. Restriction fragment length polymorphisms were identified for the members of the outer membrane protein subtype 3L group, but not for the other subtypes examined. The P2 gene from each of four prototype isolates was then cloned, sequenced and compared to the previously reported sequence of the strain MinnA gene. The P2 gene from each of two isolates with the outer membrane protein subtype 3L was identical to the MinnA P2 sequence. The P2 gene from a subtype 2L isolate differed by a single nucleotide and the gene from a subtype 6U isolate differed by 13 nucleotides. Thus, the P2 protein is highly conserved among type b isolates.     

Human antibody response to outer membrane proteins and fimbriae of Haemophilus influenzae type b

We investigated the prevalence of antibodies in childrens' sera directed against outer membrane proteins (OMP) and fimbriae of Haemophilus influenzae type b. Invasive isolates of H. influenzae type b were enriched for fimbriae production; OMP and fimbriae were resolved by SDS-PAGE. After blotting to nitrocellulose, the proteins were incubated with homologous patient sera or with sera from healthy children. IgG antibodies bound to OMP were detected by immunoperoxidase staining. Immunoblotting was also performed using purified, nondenatured fimbriae as antigen. Nine of the 10 patients studied had antibodies in the acute serum directed against one or more of the OMP. Neither the acute nor the convalescent serum of the remaining patient contained antibodies against OMP. Antibodies against a greater number of OMP were present in the convalescent serum, in comparison to the acute serum, in 4 of the 10 patients. Five of 10 patients had antibodies against the purified fimbriae of an unrelated invasive isolate in either the acute or the convalescent serum. Acute sera from patients more frequently contained antibodies directed against OMP 60K (p less than or equal to 0.01) and OMP 51K (p less than or equal to 0.003) compared with the sera of healthy controls. In contrast, the sera of healthy children more frequently contained antibodies directed against OMP 40K (p less than or equal to 0.04). Sera from both patients and controls contained antibodies against commensal Haemophilus. We conclude that although antibodies against OMP are commonly present in healthy children, antibodies against certain OMP may be markers for susceptibility or protection.     

A major outer-membrane protein functions as a porin in Haemophilus influenzae

Porins are pore-forming outer-membrane proteins which serve as a non-specific pathway for the entry of hydrophilic molecules into Gram-negative bacteria. We studied four strains of Haemophilus influenzae that had decreased permeability to chloramphenicol associated with diminished quantities of a 40 kDa major outer-membrane protein. Isogenic pairs of organisms containing and lacking this protein were compared. The latter strains grew more slowly and were less permeable to sucrose and raffinose. They were also more resistant to multiple hydrophilic antibiotics than an isogenic strain containing the 40 kDa protein and were less permeable to penicillin G and chloramphenicol. We conclude that the 40 kDa outer-membrane protein functions as a porin in H. influenzae.     

b型流感嗜血杆菌结合疫苗中载体蛋白质的效力研究

考察b型流感嗜血杆菌(Hib)结合疫苗的载体蛋白质—破伤风类毒素(TT)的免疫原性,为百白破与Hib四联疫苗中TT的使用提供参考。方法:将小鼠随机分为四组,分别注射Hib结合疫苗、Hib与百白混合疫苗、Hib与百白破混合疫苗、破伤风类毒素,比较它们在NIH小鼠体内所诱导产生的特异性抗体水平,并对这四组疫苗进行破伤风效力保护试验。结果表明,破伤风类毒素组,Hib与百白破混合疫苗组刺激的TT抗体水平远大于Hib结合疫苗组和Hib与百白混合疫苗组。效力试验虽四组间没有差异,只说明一定的抗体量就可以对小鼠提供保护。认为Hib结合疫苗中的载体蛋白并不能替代百白破疫苗中的破伤风类毒素。     

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.     

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.     

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.     

Structure and function of Hib pili from Haemophilus influenzae type b

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 A in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 A along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.