Regulation of Virulence Genes in Bordetella pertussis

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

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 (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). Virulence genes include those encoding adhesins, such as fhaB (filamentous hemagglutinin), 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+ 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, 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 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 Bvg- mode genes that could be tested for function. To expand this work we used our RNA-seq datasets 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 developed to surpass the limitations of previous bacterial sRNA search programs. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg- mode specific), of which 90 were previously unreported. We have now focused on one particular sRNA, BpsA (previously called S17), an Hfq-dependent sRNA whose level increases dramatically in the virulence (Bvg+) mode. We have demonstrated that BpsA is generated by transcription from a strong, constitutive promoter for RNA polymerase containing the primary sigma factor. This transcription yields a long, unstable form of 190 nucleotides (nts) that is processed by RNase E to generate a shorter, more stable form (BpsA-S) of 67 nts. Using RNA-seq and RT-qPCR, we have identified 92 genes whose expression significantly increases in the absence of BpsA. Of these genes, 70 contain sequences at/near their ribosome binding sites that are complementary to single-stranded (ss) regions (Sites 1 or 2) of BpsA-S. The identified genes encode transcriptional regulators, multiple transporters, and various metabolic enzymes. Using a lacZ translational reporter system, we found that BpsA-S directly represses one of these genes, BP2158, a sigma54-dependent transcriptional regulator. Sigma54 is an alternative sigma factor that is used under certain conditions and is known to be important for bacterial pathogenesis. Our results suggests that a sigma54 regulon active in the Bvg- mode is repressed by BpsA in the Bvg+ mode. Furthermore, our bioinformatics analyses have indicated that the BpsA-S region containing Sites 1 and 2 is 100% conserved throughout various Betaproteobacteria species and the BpsA-S target sites of homologs of the B. pertussis target genes are conserved in many cases as well. We speculate that BpsA-S regulation represents a highly conserved process that fine-tunes gene expression in the Bvg+ mode of B. pertussis and perhaps under other conditions in other bacteria.