The BvgAS Regulon of <i>Bordetella pertussis</i>

Abstract

Nearly all virulence factors in <i>Bordetella pertussis</i> 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 (<i>vag</i>s) are expressed [Bvg(+) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (<i>vrg</i>s) 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 <i>B. pertussis</i> 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 <i>kdpED</i>), multiple other transcriptional regulators, and the extracytoplasmic function (ECF) sigma factor <i>brpL</i>, 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 <i>in vitro</i> transcription, we demonstrate that the promoter for <i>brpL</i> is directly activated by BvgA~P. BvgA-FeBABE cleavage reactions identify BvgA~P binding sites centered at positions -41.5 and -63.5 in <i>bprL</i> Most importantly, we show for the first time that genes for multiple and varied metabolic pathways are significantly upregulated in the <i>B. pertussis</i> 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 <i>vrg</i>s that can be tested for function.<b>IMPORTANCE</b> Within the past 20 years, outbreaks of whooping cough, caused by <i>Bordetella pertussis</i>, 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 <i>B. pertussis</i> growth and infection. The two-component system BvgA (response regulator)/BvgS (histidine kinase) is the master regulator of <i>B. pertussis</i> virulence genes. We report here the first RNA-seq analysis of the BvgAS regulon in <i>B. pertussis</i>, 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.