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Principal Investigator: DEBORAH M HINTON
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2024
Award: $395,674
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). 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, S17, an Hfq-dependent sRNA whose level increases dramatically in the virulence (Bvg+) mode. We have demonstrated that S17 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 (S17S) of 67 nts. Using RNA-seq and RT-qPCR, we have identified 92 genes whose expression significantly increases in the absence of S17. 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 S17S. The identified genes encode transcriptional regulators, multiple transporters, and various metabolic enzymes. Using a lacZ translational reporter system, we found that S17S 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 S17 in the Bvg+ mode. Furthermore, our bioinformatics analyses have indicated that the S17S region containing Sites 1 and 2 is 100% conserved throughout various Betaproteobacteria species and the S17S target sites of homologs of the B. pertussis target genes are conserved in many cases as well. We speculate that S17S 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.
Terms: <Amino Acid Channel><Amino Acid Transport Systems><Amino Acid Transporter><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><Apical><B pertussis><B pertussis infection><B. pertussis><B. pertussis infection><B. pertussis vaccine><Bacteria><Bacterial Adhesins><Bacterial Infections><Base Pairing><Betaproteobacteria><Binding Sites><Bio-Informatics><Bioinformatics><Bordetella pertussis><Bordetella pertussis infection><Bordetella pertussis vaccine><Chaperone><Combining Site><Communicable Diseases><Computer Analysis><Cytoplasm><DNA-Dependent RNA Polymerases><DNA-Directed RNA Polymerase><Data><Data Set><Development><Disease><Disease Outbreaks><Disorder><Enzyme Gene><Enzymes><Epithelial Cells><Fatty Acids><Functional RNA><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><GeneHomolog><Genes><Genetic Transcription><Glycolates><Goals><Gram-Negative Bacteria><H Pertussis><H. Pertussis><Haemophilus pertussis><Health><Hemagglutinin><Homolog><Homologous Gene><Homologue><Incidence><Infant Mortality><Infant Mortality Total><Infection><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Kinases><LacZ><LacZ Genes><Messenger RNA><Metabolic><Metabolic Pathway><Miscellaneous Antibiotic><Molecular Chaperones><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Nucleotides><Organism><Outbreaks><Pathogenesis><Pathogenicity Factors><Pathway interactions><Pertussis><Pertussis Vaccine><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Play><Post-Transcriptional Control><Post-Transcriptional Regulation><Process><Protein Phosphorylation><RNA><RNA Expression><RNA Gene Products><RNA Polymerases><RNA Seq><RNA sequencing><RNAseq><RNase E><Reactive Site><Regulation><Regulon><Reporter><Repression><Resistance to antibiotics><Resistant to antibiotics><Reverse Transcription><Ribonucleic Acid><Ribosomes><Role><Sigma Element><Sigma Factor><Sigma Initiation Factor><Sigma Subunit><Site><Small RNA><Sugar Acids><Symptoms><System><T3SS><Testing><Time><Toxin><Transcription><Transcription Regulation><Transcriptional Control><Transcriptional Regulation><Translations><Transmission><Transphosphorylases><Type III Secretion System><Type III Secretion System Pathway><Untranslated RNA><Upper respiratory tract><Vaccine against B. pertussis><Vaccine against Bordetella pertussis><Vaccine against pertussis><Virulence><Virulence Factors><Western Europe><Whooping Cough><Work><adhesin><antibiotic drug resistance><antibiotic resistant><bacteria infection><bacterial disease><beta Proteobacteria><computational analyses><computational analysis><computer analyses><death among infants><death in first year of life><death in infancy><death in infants><developmental><fat metabolism><gene conservation><gene induction/repression><gene product><genome scale><genome-wide><genomewide><glycolic acid><glyoxylate><infant death><infant demise><infantile death><infected with B pertussis><infected with B. pertussis><infected with Burkholderia pertussis><lipid metabolism><living system><mRNA><mRNA Stability><mortality in infants><mutant><noncoding><pathogen><pathogenicity gene><pathway><phenylacetic acid><post-transcriptional gene regulation><posttranscriptional control><posttranscriptional regulation><programs><promoter><promotor><pyruvate dehydrogenase><response><ribonuclease E><sensor><social role><transcriptome sequencing><transcriptomic sequencing><transcriptomics><translation><transmission process><type 3 secretion system><upper airway tract><virulence gene><virulent gene><β-proteobacteria>