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Principal Investigator: DEBORAH M HINTON
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2022
Award: $291,528
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, 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.
We have now investigated the targets of a particular sRNA, whose expression is enhanced in the presence of BvgAP and Hfq. RNA-seq and RT-qPCR analyses of WT vs. a strain deleted for the sRNA identified several potential candidates for regulation; the ribosome binding sites upstream of these genes contain sequences that could potentially anneal to the 5 end of the sRNA. Beta-galactosidase assays using an E. coli strain, containing chromosomal B. pertussis target-lacZ fusions identified 3 transcripts, whose translation is significantly downregulated by the presence of the sRNA: BP2158 (a sigma 54 transcriptional activator), mmsB, (3-hydroxyisobutyrate dehydrogenase) and BP2642A-BP2638, an ABC transporter operon. We conclude that translational regulation of these genes by this sRNA contributes to the BvgAP-dependent virulent lifestyle.
Terms: <ABC Transport Protein><ABC Transporter Protein><ABC Transporters><ATP-Binding Cassette Transporters><Amino Acid Channel><Amino Acid Transport Systems><Amino Acid Transporter><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><Assay><B pertussis><B pertussis infection><B. pertussis><B. pertussis infection><Bacteria><Bacterial Adhesins><Bacterial Infections><Base Pairing><Binding><Binding Sites><Bioassay><Biologic Assays><Biological Assay><Bordetella pertussis><Bordetella pertussis infection><Chaperone><Combining Site><Communicable Diseases><Computer Analysis><Data><Data Set><Dataset><Dehydrogenases><Development><Disease><Disease Outbreaks><Disorder><E coli><E. coli><Epithelial Cells><Equilibrium><Escherichia coli><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><LacZ><LacZ Genes><Lead><Life Style><Lifestyle><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><Operon><Organism><Outbreaks><Oxidoreductase><Oxidoreductase Gene><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><Reactive Site><Reductases><Regulation><Regulon><Resistance to antibiotics><Resistant to antibiotics><Reverse Transcription><Ribonucleic Acid><Ribosomes><Role><Sigma Element><Sigma Factor><Sigma Initiation Factor><Sigma Subunit><Small RNA><Sugar Acids><Symptoms><System><T3SS><Techniques><Testing><Time><Toxin><Transcript><Transcription><Transcription Activator><Transcription Coactivator><Transcription Factor Coactivator><Transcription Regulation><Transcriptional Activator><Transcriptional Activator/Coactivator><Transcriptional Coactivator><Transcriptional Control><Transcriptional Regulation><Translational Regulation><Translations><Transmission><Transphosphorylases><Type III Secretion System><Type III Secretion System Pathway><Untranslated RNA><Upper respiratory tract><Vaccines><Virulence><Virulence Factors><Virulent><Western Europe><Whooping Cough><Work><adhesin><antibiotic drug resistance><antibiotic resistant><bacteria infection><bacterial disease><balance><balance function><beta-D-Galactosidase><beta-D-Galactoside galactohydrolase><beta-Galactosidase><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><infected with B pertussis><infected with B. pertussis><infected with Burkholderia pertussis><lac Z Protein><lipid metabolism><living system><mRNA><mRNA Stability><mutant><noncoding><northern hybridization><pathogen><pathway><phenylacetic acid><post-transcriptional gene regulation><posttranscriptional control><posttranscriptional regulation><programs><pyruvate dehydrogenase><response><sensor><social role><transcription co-activator><transcriptional co-activator><transcriptome sequencing><transcriptomics><transmission process><type 3 secretion system><upper airway tract><β-D-Galactosidase><β-D-Galactoside galactohydrolase><β-Galactosidase>