ABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Bordetella pertussis/pathogenicity , Fimbriae Proteins , Gene Expression Regulation, Bacterial , Transcription Factors/genetics , Animals , Bacterial Outer Membrane Proteins/genetics , CHO Cells , Cricetinae , Female , Lung/microbiology , Mice , Mice, Inbred BALB C , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Virulence , Virulence Factors, Bordetella/genetics , Virulence Factors, Bordetella/toxicityABSTRACT
Bordetella pertussis regulates expression of its virulence factors such as pertussis toxin (Ptx) via the bvg locus, which encodes a two-component system composed of a sensor protein, BvgS, and a transcription activator, BvgA. We used a ptx-lac fusion on the B. pertussis chromosome to analyse promoter activation by alteration of specific sequences upstream of and within the promoter. Our data demonstrate that a pair of heptanucleotide inverted repeats separated by a turn of the DNA helix within the upstream repeat region (centred around nucleotide -136.5) are crucial cis-activating elements, and probably represent the initial BvgA-binding site. In addition, we demonstrate that the sequence between these repeats and the promoter plays a role in activation. Our data are most consistent with a model of co-operative binding of BvgA dimers to this intervening region and interaction with RNA polymerase at the promoter to activate ptx transcription. In the core promoter region both the non-consensus 21 bp spacing and the specific sequence between the -35 and -10 elements are crucial for promoter activity.
