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Principal Investigator: DEBORAH M HINTON
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2019
Award: $410,289
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
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.
Terms: <Amino Acid Channel><Amino Acid Transport Systems><Amino Acid Transporter><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><B pertussis><B. pertussis><Bacteria><Bacterial Adhesins><Bacterial Infections><Base Pairing><Binding><Bordetella pertussis><Chaperone><Communicable Diseases><Computer Analysis><Data><Data Set><Dataset><Development><Disease><Disease Outbreaks><Disorder><Epithelial Cells><Equilibrium><Fatty Acids><Functional RNA><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><Genes><Genetic Transcription><Glycolates><Goals><Gram-Negative Bacteria><H Pertussis><H. Pertussis><Haemophilus pertussis><Health><Incidence><Infant Mortality><Infant Mortality Total><Infection><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Kinases><Lead><Messenger RNA><Metabolic><Metabolic Pathway><Miscellaneous Antibiotic><Molecular><Molecular Chaperones><Molecular Interaction><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Northern Blotting><Northern Blottings><Organism><Outbreaks><Pathogenesis><Pathogenicity Factors><Pathway interactions><Pb element><Pertussis><Pertussis Vaccine><Phosphotransferase Gene><Phosphotransferases><Play><Post-Transcriptional Control><Post-Transcriptional Regulation><Process><Proteins><RNA><RNA Expression><RNA Gene Products><RNA Seq><RNA blot analysis><RNA blot hybridization><RNA blotting><RNA sequencing><RNAseq><Regulation><Regulon><Resistance to antibiotics><Resistant to antibiotics><Reverse Transcription><Ribonucleic Acid><Role><Sigma Element><Sigma Factor><Sigma Initiation Factor><Sigma Subunit><Small RNA><Sugar Acids><Symptoms><System><Techniques><Testing><Time><Toxin><Transcription><Transcription Regulation><Transcriptional Control><Transcriptional Regulation><Translations><Transmission><Transphosphorylases><Untranslated RNA><Upper respiratory tract><Vaccines><Virulence><Virulence Factors><Western Europe><Whooping Cough><Work><adhesin><antibiotic drug resistance><antibiotic resistant><bacteria infection><bacterial disease><balance><balance function><computational analyses><computational analysis><computer analyses><developmental><fat metabolism><gene product><genome scale><genome-wide><genomewide><glycolic acid><glyoxylate><heavy metal Pb><heavy metal lead><lipid metabolism><living system><mRNA><mRNA Stability><mutant><noncoding><northern hybridization><pathogen><pathway><phenylacetic acid><posttranscriptional control><posttranscriptional regulation><programs><pyruvate dehydrogenase><response><sensor><social role><transcriptome sequencing><transcriptomics><transmission process>