Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin.

To identify immunologically important domains on filamentous hemagglutinin (FHA), a Bordetella pertussis protein included in new acellular pertussis vaccines (ACPVs), a series of monoclonal antibodies, sera from infants vaccinated with ACPVs or whole cell pertussis vaccine (WCPV), and sera from patients with pertussis were analyzed by immunoblots containing FHA fragments and recombinant FHA proteins. Immunodominant domains located at the COOH-terminus of FHA (type I domain) and near the NH2-terminus (type II domain) were defined by the reactivity with monoclonal antibodies. The sera from patients with pertussis and sera from infants vaccinated with WCPV or with 6 different investigational ACPVs specifically recognized well-defined regions within the type I and type II domains. Identification of these prominent immunologic epitopes on FHA should be useful for the construction of more well-defined pertussis vaccines and for the interpretation of human serologic responses, which may correlate with efficacy of pertussis vaccines.

Heparin-inhibitable lectin activity of the filamentous hemagglutinin adhesin of Bordetella pertussis.

Bordetella pertussis, the etiologic agent of whooping cough, produces an outer membrane-associated filamentous hemagglutinin (FHA) which is the major adhesin of this organism. FHA exhibits a lectin-like activity for heparin and dextran sulfate. By using in vitro adherence assays to cultured epithelial cells, the attachment of B. pertussis was reduced in the presence of sulfated polysaccharides such as heparin and dextran sulfate but not in the presence of dextran, indicating the crucial role of polysaccharide sulfation. In addition, inhibition of cellular sulfation by chlorate treatment of the cells resulted in a reduction of B. pertussis adherence, suggesting that epithelial cell surface-exposed sulfated glycoconjugates may serve as receptors for the microorganism. B. pertussis mutant strains deficient in FHA production expressed residual adherence that was no longer inhibited by sulfated polysaccharides. In addition, purified FHA displayed heparin-inhibitable binding to epithelial cells. Mapping experiments of the heparin-binding site of FHA indicated that this site is different from the RGD site and the recently proposed carbohydrate-binding site involved in the interaction of FHA with lactosylceramide. This result demonstrates that FHA contains at least three different binding sites, a feature unusual for bacterial adhesions but similar to features of eukaryotic adhesins and extracellular matrix proteins.

Inhibition of Bordetella pertussis filamentous hemagglutinin-mediated cell adherence with monoclonal antibodies.

Filamentous hemagglutinin (FHA), a 220-kDa protein located on the surface of Bordetella pertussis, is one of the major cell adhesins of this bacterium. We have produced three hybridoma cell lines that express monoclonal antibodies (mAbs) against FHA: X3C, X3E and X4B. The anti-FHA mAbs X3C and X3E reacted with 220-kDa and 98-kDa FHA protein bands on Western blots. The mAb X4B, which reacted with FHA in ELISA, did not bind to FHA in a Western blot assay. All three mAbs seemed to be directed to the same epitope or to epitopes in close proximity as suggested by competition ELISAs. All three mAbs were able to inhibit the adherence of Chinese hamster ovary cells to purified FHA, and they could also inhibit the FHA-mediated agglutination of goose red blood cells. The attachment of B. pertussis to epithelial cell monolayers was inhibited by the mAb X3C. These antibodies are very useful probes to identify the presence of FHA in bordetellae species and in clinical reagents such as pertussis vaccines, and to characterize the functional domains of this important bacterial adhesin.

Comparative roles of the Arg-Gly-Asp sequence present in the Bordetella pertussis adhesins pertactin and filamentous hemagglutinin.

Pertactin and filamentous hemagglutinin (FHA), proteins present on the surface of the gram-negative organism Bordetella pertussis, have been shown to contain the putative cell-binding sequence arginine-glycine-aspartic acid (RGD) and to promote eukaryotic cell attachment. The attachment of epithelial cells to purified pertactin and the entry of B. pertussis into human HeLa cells are both inhibited by an RGD-containing peptide derived from the pertactin sequence. In contrast, an RGD-containing peptide derived from the FHA sequence has no effect on either the attachment of epithelial cells to purified FHA or the entry of B. pertussis into HeLa cells. Staphylococcus aureus organisms coated with pertactin or FHA, purified from B. pertussis, enter HeLa cells more efficiently than S. aureus cells coated with bovine serum albumin. The pertactin-enhanced entry of S. aureus is inhibited by 75% in the presence of the RGD peptide from pertactin, whereas the RGD peptide derived from FHA has no effect on the increased entry promoted by the pertactin-coated or by the FHA-coated S. aureus. These results indicate that the active uptake of B. pertussis by certain mammalian cells may be mediated by the interaction of the RGD site found in pertactin with eukaryotic cell surface receptors.

Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis.

A group of high-molecular-weight surface-exposed proteins of nontypeable Haemophilus influenzae are major targets of human serum antibody (S. J. Barenkamp and F. F. Bodor, Pediatr. Infect. Dis. J. 9:333-337, 1990). To further characterize these proteins, we cloned and sequenced genes encoding two related high-molecular-weight proteins from a prototype nontypeable Haemophilus strain. The gene encoding a 120-kDa Haemophilus protein consisted of a 4.4-kbp open reading frame, and the gene encoding a 125-kDa protein consisted of a 4.6-kbp open reading frame. The first 1,259 bp of the two genes were identical. Thereafter, the sequences began to diverge, but overall they were 80% identical, and the derived amino acid sequences showed 70% identity. A protein sequence homology search demonstrated similarity between the derived amino acid sequences of both cloned genes and the derived amino acid sequence of the gene encoding filamentous hemagglutinin, a surface protein produced by the gram-negative pathogen Bordetella pertussis. Antiserum raised against a recombinant protein encoded by the 4.6-kbp open reading frame recognized both the 120- and the 125-kDa proteins in the prototype strain as well as antigenically related high-molecular-weight proteins in 75% of a collection of 125 epidemiologically unrelated nontypeable H. influenzae strains. The antiserum directed against the recombinant protein also recognized purified filamentous hemagglutinin. A murine monoclonal antibody to filamentous hemagglutinin recognized both the 120-kDa and the 125-kDa protein in the prototype strain as well as proteins identical to those recognized by the recombinant-protein antiserum in 35% of the nontypeable H. influenzae strain collection. Thus, we have identified and partially characterized a group of highly immunogenic surface-exposed proteins of nontypeable H. influenzae which are related to the filamentous hemagglutinin of B. pertussis.

Adhesion of Bordetella pertussis to sulfatides and to the GalNAc beta 4Gal sequence found in glycosphingolipids.

The adherence of the human respiratory pathogen, Bordetella pertussis, to purified glycosphingolipids was investigated using thin layer chromatography overlay assays. Both virulent and avirulent strains of B. pertussis bound to asialo GM1. The bacterium did not bind to the gangliosides GM1, GD1a, GD1b, and GT1b, nor to lactosylceramide, trihexosylceramide, globoside, or Forssman antigen. However, after treatment of the chromatography plates with sialidase, B. pertussis bound to the gangliosides GM1, GM2, GD1a, GD1b, and GT1b but not to GM3. Comparison of the oligosaccharide structures of these gangliosides suggests that the minimum sugar structure needed for avid bacterial binding is GalNAc beta 4Gal. This structure has been previously implicated as a receptor for other human respiratory pathogens (Krivan, H. C., Roberts, D. D., Ginsburg, V. (1988) Proc. Natl. Acad. Sci. U.S.A 85, 6157-6161). Virulent strains of B. pertussis also bound specifically to sulfatide. This response was dose-dependent and inhibited by the anionic polysaccharide dextran sulfate. The sulfated-sugars dextran sulfate, fucoidan, and heparin inhibited the attachment of virulent strains of B. pertussis to human WiDr cells and to hamster trachea cells indicating that sulfatides on the surface of mammalian cells may function as a receptor for B. pertussis. The occurrence of both sulfatides and asialo GM1 in human lung and trachea suggests that these glycolipids may serve as specific receptors for B. pertussis.

Construction and characterization of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane protein.

The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69. The cloned P.69 (prn) gene of B. pertussis was insertionally inactivated with a kanamycin-resistance cassette. This inactivated gene was used to construct P.69- mutants of B. pertussis by allelic exchange using plasmid pRTP1. B. pertussis P.69- strains produced normal levels of other vir-regulated factors, including adenylate cyclase. The serotype of B. pertussis, determined by Eldering and Preston typing sera and monoclonal antibodies, was also unaffected by the presence or absence of P.69. The ability of a prn mutant to adhere to and invade HEp2 cells was not significantly different from that of its parent strain. A strain containing a mutation in fhaB was significantly less adhesive and invasive than its parent, and a prn fhaB double mutant exhibited an even greater reduction in adhesiveness and invasiveness down to levels comparable with a Vir- strain. However, strains harbouring mutations in FHA and/or P.69 were able to colonize or multiply in the murine respiratory tract, although a Vir- strain was unable to survive and proliferate in the same infection model.

Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells.

A 69-kDa protein has been identified on the surface of the Gram-negative pathogen Bordetella pertussis that can elicit a protective immune response in animal models. This protein is associated with virulent strains of B. pertussis but its function has remained unclear. In this report we demonstrate that purified preparations of the 69-kDa outer membrane protein can promote the attachment of Chinese hamster ovary (CHO) cells. The interaction between the mammalian cells and this protein can be specifically inhibited by an Arg-Gly-Asp (RGD)-containing synthetic peptide that is homologous with a region found in the 69-kDa protein sequence. These studies indicate that a specific cell binding site containing an Arg-Gly-Asp sequence may be involved in the interaction of this bacterial protein with mammalian cell surfaces. To further investigate the role of this protein as a bacterial adhesin, a mutant of B. pertussis W28 that does not express the 69-kDa protein was constructed using the plasmid vector pRTP1. This mutant was 30-40% less efficient at adhering to CHO cells and to human HeLa cells than was the parent strain. These data support a role for this 69-kDa outer membrane protein in the attachment of B. pertussis to mammalian cells. We propose the name "pertactin" for this protein.