Bordetella pertussis, the causative agent of whooping cough, regulates expression of many virulence factors via a two-component signal transduction system encoded by the bvgAS regulatory locus. It has been shown by transcription activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed within 10 min following a bvgAS-inducing signal, while prn is transcribed after 1 h and ptx is not transcribed until 2 to 4 h after induction. These genes therefore represent early, intermediate, and late classes of bvg-activated promoters, respectively. Although there have been many insightful studies into the mechanisms of BvgAS-mediated regulation, the role that differential regulation of virulence genes plays in B. pertussis pathogenicity has not been characterized. We provide evidence that alterations to the promoter regions of bvg-activated genes can alter the kinetic pattern of expression of these genes without changing steady-state transcription levels. In addition, B. pertussis strains containing these promoter alterations that express either ptx at an early time or fha at a late time demonstrate a significant reduction in their ability to colonize respiratory tracts in an intranasal mouse model of infection. These data suggest a role for differential regulation of bvg-activated genes, and therefore for the BvgAS regulatory system, in the pathogenicity of B. pertussis.

Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvgregulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fhais transcribed at 10 min following an inducing signal, whileptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those offha and ptx. We have identifiedcis-acting sequences necessary for expression ofprn in B. pertussis by usingprn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream ofprn to activate transcription from the promoter. Our genetic data indicate that the region critical for prnactivation extends upstream to position −84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, includingprn-lac fusions, reverse transcriptase PCR, and 5′ rapid amplification of cDNA ends, to localize and identify thebvg-dependent 5′ end of the prn transcript to the cytosine at −125 with respect to the published start site.