Eighty-kilodalton N-terminal moiety of Bordetella pertussis filamentous hemagglutinin: adherence, immunogenicity, and protective role

Abstract

Bordetella pertussis, the etiological agent of whooping cough, produces a number of factors, such as toxins and adhesins, that are required for full expression of virulence. Filamentous hemagglutinin (FHA) is the major adhesin of B. pertussis. It is a protein of approximately 220 kDa, found both associated at the bacterial cell surface and secreted into the extracellular milieu. Despite its importance in B. pertussis pathogenesis and its inclusion in most acellular pertussis vaccines, little is known about the functional importance of individual domains in infection and in the induction of protective immunity. In this study, we analyzed the role of the approximately 80-kDa N-terminal domain of FHA, designated Fha44, in B. pertussis adherence, colonization, and immunogenicity. Although Fha44 contains the complete heparan sulfate-binding domain, it is not sufficient for adherence to epithelial cells or macrophages. It also cannot replace FHA during colonization of the mouse respiratory tract. Infection with a B. pertussis strain producing Fha44 instead of FHA does not induce anti-FHA antibodies, whereas such antibodies can readily be induced by intranasal administration of purified Fha44. In addition, mice immunized with purified Fha44 were protected against challenge with wild-type B. pertussis, indicating that Fha44 contains protective epitopes. Compared to FHA, Fha44 is much smaller and much more soluble and is therefore easier to purify and to store. These advantages may perhaps warrant considering Fha44 for inclusion in acellular pertussis vaccines.

FIG. 1.

Lung colonization by B. pertussis. OF1 mice were infected i.n. either with B. pertussis BPSM (solid squares) or BPSA90 (open squares) (A) or with BPRA (solid triangles), BPSA87 (solid circles), or BPDR (open circles) (B). At the indicated time points, mice were sacrificed, and viable bacteria present in the lungs were counted. Four mice were analyzed per time point for each group.

FIG. 2.

Adherence of B. pertussis to cells of two different cell lines. Human pulmonary epithelial cells (A549) and murine alveolar macrophages (MH-S) were incubated with the indicated [³⁵S]-labeled B. pertussis strains for 1 h 30 min at a multiplicity of infection of 20. After a wash, adherence was estimated by scintillation counting. Results are expressed as percentages of counts per minute present in the inoculum. Data are averages and standard deviations from quadruplicate experiments. ∗, P < 0.05 relative to BPSM values.

FIG. 3.

Protection against B. pertussis colonization. Mice were infected i.n. with B. pertussis BPSM after i.n. immunization with either purified Fha44, FHA, PT, FHA plus PT, or Fha44 plus PT, as indicated (A), or after i.v. administration of antisera from mice immunized with either PBS, PT, FHA plus PT, or Fha44 plus PT (B). Five days after infection, the mice were sacrificed, and the viable bacteria present in the lungs were counted. Values shown are means ± standard deviations for at least four mice per group. ∗, P < 0.05 relative to the PBS control. The dashed line in panel A represents the limit of detection of the number of CFU present in the lungs.

FIG. 4.

Protection against B. pertussis colonization by using CT plus CTB as an adjuvant. Mice were infected i.n. with B. pertussis BPSM after i.n. immunization with either purified CT plus CTB, Fha44, FHA, or Fha44 plus CT plus CTB, as indicated. Two days after infection, mice were sacrificed, and viable bacteria in the lungs were counted. Values shown are means ± standard deviations for at least four mice per group. ∗, P < 0.05 relative to the PBS control.