Combined RNAseq and ChIPseq Analyses of the BvgA Virulence Regulator of Bordetella pertussis

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.

Keywords: Bordetella pertussis; BvgA; ChIPseq; RNAseq; response regulator.

FIG 1

Venn diagrams showing the numbers of overlapping genes between BPSM Mg and BPSMΔBvgA. vags(-3) (A) and vags(-2) (C) are genes that present Log₂FCs in transcription of less than −3 and −2, respectively, compared to that in BPSM. vrgs(+3) (B) and vrgs(+2) (D) are genes that present Log₂FCs in transcription of >3 and >2, respectively, compared to that in BPSM.

FIG 2

Transcription profiling, RACE, and qRT PCR analysis on the bvgA promoter region. (A) RPKM quantification in BPSM, modulated BPSM (BPSM Mg), and BPSMΔBvgAfor bvgS, bvgA, and fhaB. bvgA13AA corresponds to the region coding for the 13 first amino acids of BvgA. RPKM standard deviations (SD)s are shown. (B) Schematic representation of the B. pertussis fhaB-bvgAS promoter region. P1, P2, and P3 represent the described bvgA promoters. qRT bvgA P1 and P2 depict the regions used to quantify the promoter strength of P1 and P2, respectively, by qRT PCR. (C) Mapping of the RACE reads obtained with the BvgA RACE primer on BPSM, modulated BPSM (BPSM Mg), and BPSMΔBvgA RNA. Single reads mapping in the forward direction are in green. Single reads mapping in the reverse direction are red. Any mismatches between the reads and the reference sequence are shown as colored dots. (D) Quantitative RT-PCR analysis of P1 and P2 promoter regions of bvgA. mRNA transcripts corresponding to the bvgA promoters P1 and P2, as shown in B, were amplified by qRT-PCR using a bvgA-specific reverse primer and a primer corresponding to either P1 or P2 (see panel B). Expression of bvgA P1 and P2 transcripts is presented relative to the level of expression of the housekeeping gene bp3416.

FIG 3

Graphical representation of the different BvgA-binding sites with respect to identified BvgA-regulated genes on the B. pertussis genome. Numbers correspond to the numbers of BvgA-binding sites in each category.

FIG 4

MEME analysis of motifs identified in the ChIPseq peaks. (A) Motif found using the 148 putative BvgA-binding sites. (B) Motif found using the 91 putative BvgA-binding sites located in promoter regions.