The BvgAS Regulon of Bordetella pertussis

Abstract

Nearly all virulence factors in Bordetella pertussis are activated by a master two-component system, BvgAS, composed of the sensor kinase BvgS and the response regulator BvgA. When BvgS is active, BvgA is phosphorylated (BvgA~P), and virulence-activated genes (vags) are expressed [Bvg(+) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (vrgs) are induced [Bvg(-) mode]. Here, we have used transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR) to define the BvgAS-dependent regulon of B. pertussis Tohama I. Our analyses reveal more than 550 BvgA-regulated genes, of which 353 are newly identified. BvgA-activated genes include those encoding two-component systems (such as kdpED), multiple other transcriptional regulators, and the extracytoplasmic function (ECF) sigma factor brpL, which is needed for type 3 secretion system (T3SS) expression, further establishing the importance of BvgA~P as an apex regulator of transcriptional networks promoting virulence. Using in vitro transcription, we demonstrate that the promoter for brpL is directly activated by BvgA~P. BvgA-FeBABE cleavage reactions identify BvgA~P binding sites centered at positions -41.5 and -63.5 in bprL Most importantly, we show for the first time that genes for multiple and varied metabolic pathways are significantly upregulated in the B. pertussis Bvg(-) mode. These include genes for fatty acid and lipid metabolism, sugar and amino acid transporters, pyruvate dehydrogenase, phenylacetic acid degradation, and the glycolate/glyoxylate utilization pathway. Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new vrgs that can be tested for function.IMPORTANCE Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due, at least in part, to the introduction of a less effective acellular vaccine in the 1990s. It is crucial, then, to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA (response regulator)/BvgS (histidine kinase) is the master regulator of B. pertussis virulence genes. We report here the first RNA-seq analysis of the BvgAS regulon in B. pertussis, revealing that more than 550 genes are regulated by BvgAS. We show that genes for multiple and varied metabolic pathways are highly regulated in the Bvg(-) mode (absence of BvgA phosphorylation). Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence.

Keywords: BvgAS regulon; RNA polymerase; RNA-seq; brpL; pertussis.

FIG 1

Relative expression of selected genes of transcriptional regulators positively regulated by BvgA~P. Values are shown as fold difference using the 2^−ΔΔCT method (65). Error bars indicate standard deviations among three independent biological replicates for each condition with more than two different trials. Blue bars, FD of WT without MgSO₄ versus WT with MgSO₄; red bars, FD of WT without MgSO₄ versus ΔbvgAS strain without MgSO₄. Student’s t test was used for statistical analysis, and genes showing significant differences between WT without MgSO₄ and WT with MgSO₄ or between WT without MgSO₄ and ΔbvgAS strain without MgSO₄ are labeled as *** (P < 0.001), ** (P < 0.01), or * (P < 0.05). “ns” represents statistically nonsignificant.

FIG 2

Relative expression of selected genes involved in metabolic pathways negatively affected by BvgA~P, as measured by RT-qPCR. Values are shown as fold difference using the 2^−ΔΔCT method (65). Error bars indicate standard deviations among three independent biological replicates for each condition with more than two different trials. Blue bars, FD of WT without MgSO₄ versus WT with MgSO₄; red bars, FD of WT without MgSO₄ versus ΔbvgAS strain without MgSO₄. Bracketed genes are located in the same locus of the genome. Student’s t test was used for statistical analysis, and genes showing significant differences between WT without MgSO₄ and WT with MgSO₄ or between WT without MgSO₄ and ΔbvgAS strain without MgSO₄ are labeled as *** (P < 0.001), ** (P < 0.01), or * (P < 0.05).

FIG 3

P_brpL is directly activated by BvgA~P. (A) Denaturing polyacrylamide gels showing RNA obtained after in vitro transcription of the indicated templates using (as indicated) B. pertussis RNAP alone (−), RNAP plus BvgA (+), or RNAP plus BvgA~P (P). The expected transcripts (nucleotides) are indicated. (B) Primer extension analysis of in vitro RNA to identify the start site of P_brpL. A denaturing gel of primer extension products is shown. Left lanes are sequencing ladders; right lanes show primer extension products for RNA obtained in the absence of BvgA (−), the presence of BvgA~P (P), or the presence of nonphosphorylated BvgA (+). (C) Sequence of the brpL promoter, determined from primer extension analyses in panel A, and of the promoters for BP1005 and BP3441, determined from transcript length in in vitro transcription in panel B. Assigned −10 and −35 elements (BP1005 and BP3441) are in blue; +1 is in red.

FIG 4

FeBABE cleavage sites for P_brpL and P_fhaB. (A) Denaturing gels showing products of FeBABE cleavage using complexes formed with P_brpL (left) or P_fhaB (right), ³²P end labeled on either the template or the nontemplate strand, B. pertussis RNAP, and (as indicated) either phosphorylated BvgA^T194C-FeBABE or phosphorylated BvgA^V148C-FeBABE. The G+A lanes correspond to DNA ladders generated by Maxam-Gilbert sequencing with the positions relative to the transcription start sites indicated (36). Arrowheads denote sites of cleavage. (B) Sequences of P_brpL and P_fhaB with the cut sites generated by BvgA^T194C-FeBABE (pink arrowheads) and by BvgA^V148C-FeBABE (purple arrowheads). BvgA binding sites are boxed in orange; the −10 regions are shown and boxed in blue. (An alternative −10 element for P_fhaB is indicated in the dashed purple box.) Transcription start sites (+1) are in red. Numbers above the inverted red arrows indicate the predicted score for BvgA binding half-sites in each promoter according to the algorithm reported by Merkel et al. (67). For instance, the sequence of the perfect inverted repeat is TAGGAAATTTCCTA, whose score is given as “0.”