Combined transcriptomic and ChIPseq analyses of the Bordetella pertussis RisA regulon

Abstract

The regulation of Bordetella pertussis virulence is mediated by the two-component system BvgA/S, which activates the transcription of virulence-activated genes (vags). In the avirulent phase, the vags are not expressed, but instead, virulence-repressed genes (vrgs) are expressed, under the control of another two-component system, RisA/K. Here, we combined transcriptomic and chromatin immunoprecipitation sequencing (ChIPseq) data to examine the RisA/K regulon. We performed RNAseq analyses of RisA-deficient and RisA-phosphoablative B. pertussis mutants cultivated in virulent and avirulent conditions. We confirmed that the expression of most vrgs is regulated by phosphorylated RisA. However, the expression of some, including those involved in flagellum biosynthesis and chemotaxis, requires RisA independently of phosphorylation. Many RisA-regulated genes encode proteins with regulatory functions, suggesting multiple RisA regulation cascades. By ChIPseq analyses, we identified 430 RisA-binding sites, 208 within promoter regions, 201 within open reading frames, and 21 in non-coding regions. RisA binding was demonstrated in the promoter regions of most vrgs and, surprisingly, of some vags, as well as for other genes not identified as vags or vrgs. Unexpectedly, many genes, including some vags, like prn, brpL, bipA, and cyaA, contain a BvgA-binding site and a RisA-binding site, which increases the complexity of the RisAK/BvgAS network in B. pertussis virulence regulation.IMPORTANCEThe expression of virulence-activated genes (vags) of Bordetella pertussis, the etiological agent of whooping cough, is under the transcriptional control of the two-component system BvgA/S, which allows the bacterium to switch between virulent and avirulent phases. In addition, the more recently identified two-component system RisA/K is required for the expression of B. pertussis genes, collectively named vrgs, that are repressed during the virulent phase but activated during the avirulent phase. We have characterized the RisA/K regulon by combined transcriptomic and chromatin immunoprecipitation sequencing analyses. We identified more than 400 RisA-binding sites. Many of them are localized in promoter regions, especially vrgs, but some were found within open reading frames and in non-coding regions. Surprisingly, RisA-binding sites were also found in promoter regions of some vags, illustrating the previously underappreciated complexity of virulence regulation in B. pertussis.

Keywords: Bordetella pertussis; ChIPseq; RNAseq; RisA.

Fig 1

Regulation of genes by RisA and modulation. Volcano plots showing the regulation of the genes in nmBPSMΔrisA vs nmBPSM (A), nmBPSMrisA^D60N vs nmBPSM (B), mBPSM vs nmBPSM (D), mBPSMrisAD60N vs mBPSM (E), and mBPSMΔrisA vs mBPSM (F). The vertical gray bars represent the cutoff used (Log₂FC −3 or +3). Genes were clusterized and are presented in different colors according to their regulation presented on the heat map (C). On the heat map, the first column (black boxes) shows the presence of RisA-binding sites in the promoter regions. The following columns present heat maps of the gene regulation comparing nmBPSMΔrisA vs nmBPSM (nmDRisA vs nmWT), nmBPSMrisA^D60N vs nmBPSM (nmRisA^D60N vs nmWT), mBPSM vs nmBPSM (mWT vs nmWT), mBPSMΔrisA vs mBPSM (mDRisA vs mWT), and mBPSMrisA^D60N vs mBPSM (mRisA^D60N vs mWT).

Fig 2

Regulated putative sRNA identified by B. pertussis BPSM RNAseq analysis. Transcriptional start and stop sites are the first and last nucleotides of the detected putative sRNA relative to the B. pertussis Tohama I BX470248 genome annotation. Strands “+” or “−” represent forward and reverse directions, respectively, to the orientation of the B. pertussis Tohama I BX470248 genome annotation. Synonyms correspond to previously described sRNA. RisA binding corresponds to this study, while BvgA binding is from Coutte et al. (2). Log₂FC corresponds to the Log₂ fold change of the RPKM of the detected putative sRNA in the tested conditions. nt, nucleotides.

Fig 3

Regulation of genes coding for putative B. pertussis regulators. Genes and gene products correspond to the B. pertussis Tohama I BX470248 genome annotation. Log₂FC corresponds to the Log₂ fold change of the RPKM of the gene in the tested conditions. RisA binding corresponds to this study, while BvgA binding is from Coutte et al. (2).

Fig 4

Summary of ChIPseq analysis of the B. pertussis RisA regulon. Putative promoter regions are located between two ORFs orientated in opposite directions. Non-coding regions are not annotated in the B. pertussis Tohama I BX470248 genome. Inside means within annotated ORF of the B. pertussis Tohama I BX470248 genome. vags present a Log₂FC < −3 in the mBPSM RNAseq analysis. vrgs present a Log₂FC > 3 in the mBPSM RNAseq analysis. No vag No vrg are genes not identified as vag or vrg in the RNAseq analysis. RisA downregulated genes present a Log₂FC < −3 in the nmBPSMΔrisA RNAseq analysis. RisA upregulated genes present a Log₂FC of >3 in the nmBPSMΔrisA RNAseq analysis. Bvgⁱ corresponds to bipA, not regulated by modulation under the conditions tested here but requiring BvgA, as described by Coutte et al. (5).

Fig 5

Localization of the RisA-binding sites within ORFs. ChIPseq peaks are depicted according to the localization within the ORF and are expressed as percentages of the total length of the corresponding ORF.

Fig 6

5′ RACE analysis of the prn promoter region. Schematic representation of the B. pertussis prn promoter region. The prn ORF start site is depicted by the yellow arrow, as annotated in the B. pertussis Tohama I BX470248 genome. Position of the TSS of prn, indicated by the blue arrow, was described by Kinnear et al. (11). Position of the BvgA-binding site, indicated by the blue box, was described in reference (2). Position of the RisA-binding site, indicated by the yellow box, is described in this study. Mapping of the RACE reads obtained with BPSM, BPSMΔrisA, and BPSMrisA^D60N in modulating and non-modulating conditions are depicted in blue.

Fig 7

MEME analysis of the RisA ChIPseq peaks. (A) Motif found using the 430 RisA-binding sites. The number of sites contributing to the construction of the motif = 391, E-value 2.6 e⁻¹⁰⁶, and 430 sequences used. (B) Number of RisA motif occurrences in the 430 RisA ChIPseq peaks according to the localization. Putative promoter regions are located between two ORFs orientated in opposite directions. Non-coding regions are not annotated in the B. pertussis Tohama I BX470248 genome. Inside means within annotated ORF of the B. pertussis Tohama I BX470248 genome. nt, nucleotides.

Fig 8

Proposed model of gene regulation by BvgA and RisA in B. pertussis. The kinases BvgS and RisK phosphorylate BvgA and RisA, respectively. In avirulent conditions, bvgR is not expressed, and therefore c-di-GMP levels are elevated and serve as a co-factor of RisA, which regulates the expression of several clusters of genes directly by binding to the corresponding promoter region, or indirectly, via a regulation cascade. Solid lines represent direct regulation, as demonstrated by the ChIPseq results, and dashed lines represent indirect regulation, as demonstrated by the ChIPseq and RNAseq results.