Characterization of differential regulation and its role in the pathogenesis of Bordetella pertussis

Abstract

Bordetella pertussis, the causative agent of whooping cough, regulates expression of its 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 minutes following an inducing signal, while ptx is not transcribed until 2-4 hours later. We present data indicating that, while fim2 is transcribed early similar to fha, prn is transcribed after 1 hour, an intermediate time. We have identified cis-acting sequences necessary for transcription of prn in B. pertussis using prn-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 of prn to activate transcription from the promoter. Our genetic data indicate that the region critical for prn activation extends upstream to -84. We used a number of methods including prn-lac fusion analysis, RT-PCR, and 5' RACE to identify the bvg-dependent 5' end of the prn transcript as the cytosine at -125 with respect to the previously published transcription start site. We have also provided evidence that there may be an inhibitory effect of the binding of high concentrations of BvgA at the prn promoter. 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 pathogenesis has not been characterized. We provide evidence that suggests 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 ptx promoter alterations that express ptx at an early time, demonstrate a significant reduction in the ability to colonize respiratory tracts in an intranasal mouse model of infection. The colonization defect can be mimicked by the maintenance of the wild type B. pertussis inoculum at 37°C prior to inoculation, thus suppressing modulation of the BvgAS system and down-regulation of ptx expression. This data suggests a role for differential regulation of bvg-activated genes, and subsequently for the BvgAS regulatory system, in the pathogenesis of B. pertussis.