Identification and characterization of BipA, a Bordetella Bvg‐intermediate phase protein

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Abstract
The Bordetella BvgAS sensory transduction system has traditionally been viewed as controlling a transition between two distinct phenotypic phases: the Bvg+ or virulent phase and the Bvg− or avirulent phase. Recently, we identified a phenotypic phase of Bordetella bronchiseptica that displays reduced virulence in a rat model of respiratory infection concomitant with increased ability to survive nutrient deprivation. Characterization of this phase, designated Bvg‐intermediate (Bvgi), indicated the presence of antigens that are maximally, if not exclusively, expressed in this phase and therefore suggested the existence of a previously unidentified class of Bvg‐regulated genes. We now report the identification and characterization of a Bvgi phase protein, BipA (Bvg‐intermediate phase protein A), and its structural gene, bipA. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis indicates that bipA is expressed maximally under Bvgi phase conditions and thus represents the first identified Bvgi phase gene. bipA encodes a 1578‐amino‐acid protein that shares amino acid sequence similarity at its N‐terminus with the proposed outer membrane localization domains of intimin (Int) of enteropathogenic and enterohaemorrhagic Escherichia coli and invasin (Inv) of Yersinia spp. Although not apparent at the amino acid level, BipA is also similar to Int and Inv in that the proposed membrane‐spanning domain is followed by several 90‐amino‐acid repeats and a distinct C‐terminal domain. Localization studies using an antibody directed against the C‐terminus of BipA indicated that its C‐terminus is exposed on the bacterial cell surface. Western blot analysis with this same antibody indicated that BipA homologues are expressed in Bvgi phase Bordetella pertussis and Bordetella parapertussis. Comparison of a ΔbipA strain with wild‐type B. bronchiseptica indicated that BipA is not required for Bvgi phase‐specific aggregative adherence to rat lung epithelial cells in vitro or for persistent colonization of the rabbit respiratory tract in vivo. However, our data are consistent with the hypothesis that BipA, and the Bvgi phase in general, play an important role in the Bordetella infectious cycle, perhaps by contributing to aerosol transmission.