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 (BvgAP), and virulence activated genes (vags) are expressed the Bvg(+) mode. When BvgS is inactive and BvgA is not phosphorylated, virulence repressed genes (vrgs) are induced the Bvg(-) mode. The Bvg(i) mode represents an intermediate state, with an intermediate concentration of BvgAP where kinase-on and kinaseoff BvgS proteins may co-exist in equilibrium. The B. pertussis virulence genes include adhesins, needed to adhere to the ciliated epithelial cells within the upper respiratory tract, and toxins, which cause the major symptoms of whooping cough disease. Several of these BvgA-activated gene products are components of the acellular pertussis vaccine used in the U.S. and Western Europe. We have used RNA-seq and RT-qPCR to define the BvgAS-dependent regulon of the virulent B. pertussis strain 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 ECF sigma factor, brpL, which is needed for Type 3 Secretion System expression, further establishing the importance of BvgAP as an apex regulator of transcriptional networks promoting virulence. Using in vitro transcription, we have demonstrated that the promoter for brpL is directly activated by BvgAP. BvgA-FeBABE cleavage reactions identify BvgAP 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 up-regulated 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. Despite extensive studies, the role of BvgAS regulation in the natural history of B. pertussis has not been clear. For Bordetella bronchiseptica, a close relative of B. pertussis that has a broad mammalian host range, the Bvg(-) mode has been shown to contribute to survival outside the host environment in laboratory experiments. Although B. bronchiseptica has never been cultured from natural environments, this provides at least a plausible rationale for BvgAS-mediated regulation in this species. B. pertussis, on the other hand, does not survive for long periods outside of the host respiratory tract, and its natural host range is limited to humans. In addition, the B. pertussis genome has undergone significant contraction and gene inactivation since its evolution from a B. bronchiseptica-like common ancestor, leading one to think of it as a degraded species. Our work is significant because at least two different rationalizations for the presence of the BvgAS system in B. pertussis have previously been put forward. One holds that the BvgAS regulatory system is an evolutionary fossil, and although it may have played an important role in optimizing gene expression in different environments in an ancestor, it simply functions now for virulence gene expression. Consequently, the Bvg(-) and Bvg(i) modes are not important. Consistent with this view is that B. pertussis does not require the Bvg(-) mode for full virulence in animal models of infection. However, even though mutants that express virulence genes constitutively can arise easily by single nucleotide changes, this environmental responsiveness has been conserved in evolution. An alternative possibility is that, while the mode of passage between hosts may have become somewhat different and much more abbreviated for B. pertussis, the Bvg(-) or Bvg(i) modes still play a role in maximizing aerosol transmission. Conceivably, this could occur by allowing bacteria to respond effectively to stresses encountered in the harsh environment of an aerosol created by coughing, by aiding in the preparation for expulsion from the host, or by aiding in the initial establishment of infection after deposition in a new host. Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence, and strongly support the idea that the Bvg(-) and BvgA(i) are important for infectivity of the disease. Our identification of new metabolic genes that are induced in the Bvg(-) mode provides a starting point for investigating how these metabolic changes help the pathogen as it proceeds through its complete life cycle.
