GSE98155
The BvgAS-dependent regulon of Bordetella pertussis
Purpose: 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 1990's. It is crucial then to understand the molecular basis of B. pertussis growth and infection.  The two-component system BvgA/BvgS is the B. pertussis master regulator that regulates the expression of various virulence genes.  Previous genome wide studies to identify the bvgAS regulon in B. pertussis have been limited to microarray analyses.  The goal of this study is to define the bvgAS regulon using NGS based RNA-seq analysis and RT-qPCR and to compare the differences with the previous microarray data. Method: To define the bvgAS regulon, transcriptomes were generated using B. pertussis Tohama I BP536 (WT) and a ?bvgAS derivative in the presence and absence of phosphorylated BvgA. Bacteria were grown either under modulating (50 mM MgSO4) conditions, in which phosphorylated BvgA is undetectable (Bvg- mode), or non-modulating (no MgSO4) conditions in which BvgA is phosphorylated (Bvg+ mode). After total RNA extraction and rRNA removal, strand-specific DNA libraries were prepared for Illumina sequencing using the ScriptSeq 2.0 kit (Illumina) in duplicates.  Libraries were sequenced using a HiSeq 2000 sequencer (Illumina; University of Buffalo Next Generation Sequencing Core Facility). Sequence reads were mapped to the reference genome (NC_002929.2) and normalized against total reads. Differential expression analyses were performed using CLC Biomedical Genomic Workbench version 3.5.4 with default parameters. Differential gene expression profiles were obtained after Empirical DGE statistical test with FDR p-value. RT–qPCR validation was performed using SYBR Green assays. Result: Using CLC Biomedical Genomic Workbench version 3.5.4, ~ 20-25 million reads per sample were mapped to the reference B. pertussis genome (NC_002929.2).  RNA-seq analyses identified >550 genes (15% of genome) that were regulated in a bvgAS-dependent manner with a fold difference = 1.6 and FDR p-value < 0.05.  RT-qPCR was used for confirmatory analyses for 80 genes.  Overall, we identified 245 genes that were positively regulated and 326 genes that were negatively regulated by bvgAS.  362 members of the bvgAS regulon were newly identified by our study.  Most importantly, our analyses indicated that the expression of dozens of transcriptional regulators increases, while the expression of multiple metabolic genes decreases in the presence of BvgA~P. Conclusion: We report here the first RNA-seq analysis of the bvgAS regulon in B. pertussis, revealing that > 550 genes are regulated by bvgAS. Our results suggest that metabolic changes in the Bvg- mode (absence of BvgA phosphorylation) may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new genes negatively regulated by bvgAS that can be tested for function. Overall design: Two strains were used to identify the bvgAS regulon: WT and an isogenic ?bvgAS derivative.  No MgSO4 or addition of 50 mM MgSO4 to the growth medium was used to generate the BvgA+ mode (presence of BvgA phosphorylation) or the Bvg- mode (absence of BvgA phosphorylation), respectively.  B. pertussis mRNA profiles were generated by deep sequencing, in duplicates, using Illumina ScriptSeq 2.0 kit.