To successfully colonize their mammalian hosts many bacteria produce multiple virulence factors that play essential roles in disease processes and pathogenesis. Some of these molecules are adhesins that allow efficient attachment to host cells, a prerequisite for successful host colonization. Bordetella spp. express a number of proteins which either play a direct role in attachment to the respiratory epithelia or exhibit similarity to previously known bacterial adhesins. One such recently identified protein is BipA. Despite similarity to intimins and invasins, its deletion from B. bronchiseptica did not result in any significant defect in respiratory tract colonization. We hypothesized the existence of a paralogous protein that could complement the function of BipA. In the studies described here, we report the identification and characterization of an ORF in B. bronchiseptica, designated as bcfA ( Bordetella colonization factor A) that is similar to bipA . We show that in contrast to maximal expression of bipA in the Bvgⁱ phase, bcfA is expressed at high levels in both the Bvg⁺ and the Bvgi phases. We have identified multiple sequence elements resembling the consensus BvgA binding site in the bcfA promoter region. Direct binding of purified BvgA to the bcfA promoter revealed differences in the DNA binding profiles of BvgA and BvgA-P to the promoter region. Utilizing an antibody raised against BcfA, we show that BcfA is localized in the outer membrane. Finally, we demonstrate that simultaneous deletion of both bipA and bcfA results in a defect in colonization of the rat trachea and that BcfA is expressed during Bordetella infectious cycle.

Based on our findings that BcfA is an outer membrane immunogenic protein and is critical for murine respiratory tract colonization, we examined its utility in inducing a protective immune response against B. bronchiseptica in a mouse model of intranasal infection. Mice vaccinated with BcfA demonstrated reduced pathology in the lungs and harbored lower bacterial burdens in the respiratory tract. Immunization with BcfA led to the generation of BcfA-specific antibodies in both the serum and the lungs and passive immunization led to the reduction of B. bronchiseptica in the trachea and the lungs. These results suggest that protection after immunization with BcfA is mediated in part by antibodies against BcfA. To further investigate the mechanism of BcfA-induced immune clearance, we examined the role of neutrophils and macrophages. Our results demonstrate that neutrophils are critical for anti-BcfA antibody-mediated clearance and that opsonization with anti-BcfA serum enhances phagocytosis of B. bronchiseptica by murine macrophages. We show that immunization with BcfA results in the production of IFN-γ and subclasses of IgG antibodies that are consistent with the induction of a Th1 type immune response. In combination, our findings suggest that mechanism of BcfA-mediated immunity involves humoral and cellular responses. Expression of BcfA is conserved among multiple clinical isolates of B. bronchiseptica. Our results demonstrate the striking protective efficacy of BcfA-mediated immunization thereby highlighting its utility as a potential vaccine candidate. These results also provide a model for the development of cell-free vaccines against B. bronchiseptica.