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 are expressed (the Bvg(+) mode). When BvgS is inactive and BvgA is not phosphorylated, virulence repressed genes are induced (the Bvg(-) mode). The Bvg(i) mode represents an intermediate state, with an intermediate concentration of BvgAP where kinase-on and kinase-off BvgS proteins may co-exist in equilibrium. Virulence genes include those encoding adhesins, such as the fimbrial subunits fim2 and fim3, which are 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 previously 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 revealed more than 550 BvgA-regulated genes, of which 353 were 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 BvgAP as an apex regulator of transcriptional networks promoting virulence. Most importantly, we showed 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 suggested that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence and identified >200 new vrgs that could be tested for function. To expand this work we have used this RNA-seq data set to conduct a genome-wide transcriptomic search for non-coding small RNAs (sRNAs) in B. pertussis. sRNAs play a crucial role in post-transcriptional regulation of gene expression in all organisms. A major class of sRNAs in bacteria regulates translation and mRNA stability by base pairing with their target mRNAs via an interaction facilitated by the RNA chaperone Hfq. In pathogens, Hfq and Hfq-dependent sRNAs regulate a wide spectrum of virulence gene expression and are involved in key steps of the infection process. To identify sRNAs in B. pertussis, WT and bvgAS- strains were grown both without MgSO4 (nonmodulating conditions, resulting in the BvgA(+) mode) and with MgSO4 (modulating conditions, resulting in the BvgA(-) mode). To process the data, we performed a computational analysis using the prokaryotic sRNA search program, ANNOgesic, which was recently developed to surpass the limitations of current bacterial sRNA search programs. We picked 20 candidates to analyze by Northern blots and Hfq-binding studies. Our study demonstrates that combining RNA-seq, ANNOgesic, and molecular techniques is a successful approach to identify various BvgAS-dependent and Hfq binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.

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