Abstract
Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS. In the virulence phase, BvgS phosphorylates BvgA, which then activates the transcription of virulence-activated genes (vags). In the avirulence phase, such as during growth in the presence of MgSO4, BvgA is not phosphorylated and the vags are not expressed. Instead, a set of virulence-repressed genes (vrgs) is expressed. Here, we performed transcriptome sequencing (RNAseq) analyses on B. pertussis cultivated with or without MgSO4 and on a BvgA-deficient Tohama I derivative. We observed that 146 genes were less expressed under modulating conditions or in the BvgA-deficient strain than under the nonmodulating condition, while 130 genes were more expressed. Some of the genes code for proteins with regulatory functions, suggesting a BvgA/S regulation cascade. To determine which genes are directly regulated by BvgA, we performed chromatin immunoprecipitation sequencing (ChIPseq) analyses. We identified 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Among the former, 32 are in BvgA-regulated putative promoter regions. Some vags, such as dnt and fhaL, contain no BvgA-binding site, suggesting indirect BvgA regulation. Unexpectedly, BvgA also bound to some vrg putative promoter regions. Together, these observations indicate an unrecognized complexity of BvgA/S biology.IMPORTANCE Bordetella pertussis, the etiological agent of whooping cough, remains a major global health problem. Despite the global usage of whole-cell vaccines since the 1950s and of acellular vaccines in the 1990s, it still is one of the most prevalent vaccine-preventable diseases in industrialized countries. Virulence of B. pertussis is controlled by BvgA/S, a two-component system responsible for upregulation of virulence-activated genes (vags) and downregulation of virulence-repressed genes (vrgs). By transcriptome sequencing (RNAseq) analyses, we identified more than 270 vags or vrgs, and chromatin immunoprecipitation sequencing (ChIPseq) analyses revealed 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Some vags, such as dnt and fhaL, do not contain a BvgA-binding site, suggesting indirect regulation. In contrast, several vrgs and some genes not identified by RNAseq analyses under laboratory conditions contain strong BvgA-binding sites, indicating previously unappreciated complexities of BvgA/S biology.
Highlights
Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS
RNAseq analyses were performed to decipher the global regulation of B. pertussis transcriptomes by BvgA according to its state of phosphorylation
RNA was isolated from the B. pertussis Tohama I derivative BPSM [9] grown in the absence or presence of 50 mM MgSO4 (BPSM Mg, referred to as the modulating condition) and from BPSMΔBvgA, a BPSM derivative carrying a genetic deletion of bvgA and in which bvgS is located directly downstream of the bvgA/S promoter
Summary
Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS. A number of studies using microarray [3,4,5,6] or, more recently, transcriptome sequencing (RNAseq) technologies [7, 8] have been carried out to define the Bvg regulon by identifying genes whose expression is down- (for the vags) or upregulated (for the vrgs) by the addition of modulators or, in BvgA/S-deficient mutants compared to that in isogenic parent strains, when grown in the absence of modulators These studies did not distinguish vags that are directly regulated by BvgA through its binding to the promoter/operator regions from those that are indirectly regulated by BvgA via a regulatory cascade involving intermediate transcription factors. We combined RNAseq and chromatin immunoprecipitation sequencing (ChIPseq) analyses to identify vags together with BvgA-binding sites on the B. pertussis chromosome as a first step to decipher the global BvgA/S regulon cascade
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