Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic
ABSTRACT Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primari...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2021-10-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/Spectrum.00044-21 |
| _version_ | 1798034072824446976 |
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| author | Kyung Moon Minji Sim Chin-Hsien Tai Kyungyoon Yoo Charlotte Merzbacher Sung-Huan Yu David D. Kim Jaehyun Lee Konrad U. Förstner Qing Chen Scott Stibitz Leslie G. Knipling Deborah M. Hinton |
| author_facet | Kyung Moon Minji Sim Chin-Hsien Tai Kyungyoon Yoo Charlotte Merzbacher Sung-Huan Yu David D. Kim Jaehyun Lee Konrad U. Förstner Qing Chen Scott Stibitz Leslie G. Knipling Deborah M. Hinton |
| author_sort | Kyung Moon |
| collection | DOAJ |
| description | ABSTRACT Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg− mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg− mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis. |
| first_indexed | 2024-04-11T20:39:12Z |
| format | Article |
| id | doaj.art-ba3b727b18e24be4a854bd8c0b732ef9 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-11T20:39:12Z |
| publishDate | 2021-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-ba3b727b18e24be4a854bd8c0b732ef92022-12-22T04:04:16ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972021-10-019210.1128/Spectrum.00044-21Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesicKyung Moon0Minji Sim1Chin-Hsien Tai2Kyungyoon Yoo3Charlotte Merzbacher4Sung-Huan Yu5David D. Kim6Jaehyun Lee7Konrad U. Förstner8Qing Chen9Scott Stibitz10Leslie G. Knipling11Deborah M. Hinton12Gene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USALaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USAGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAInstitute of Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, GermanyGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAInstitute of Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, GermanyDivision of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USADivision of Bacterial, Parasitic, and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USAGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAGene Expression and Regulation Section, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USAABSTRACT Noncoding small RNAs (sRNAs) are crucial for the posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. In the human pathogen Bordetella pertussis, which causes whooping cough, virulence is controlled primarily by the master two-component system BvgA (response regulator)/BvgS (sensor kinase). In this system, BvgA is phosphorylated (Bvg+ mode) or nonphosphorylated (Bvg− mode), with global transcriptional differences between the two. B. pertussis also carries the bacterial sRNA chaperone Hfq, which has previously been shown to be required for virulence. Here, we conducted transcriptomic analyses to identify possible B. pertussis sRNAs and to determine their BvgAS dependence using transcriptome sequencing (RNA-seq) and the prokaryotic sRNA prediction program ANNOgesic. We identified 143 possible candidates (25 Bvg+ mode specific and 53 Bvg− mode specific), of which 90 were previously unreported. Northern blot analyses confirmed all of the 10 ANNOgesic candidates that we tested. Homology searches demonstrated that 9 of the confirmed sRNAs are highly conserved among B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, with one that also has homologues in other species of the Alcaligenaceae family. Using coimmunoprecipitation with a B. pertussis FLAG-tagged Hfq, we demonstrated that 3 of the sRNAs interact directly with Hfq, which is the first identification of sRNA binding to B. pertussis Hfq. Our study demonstrates that ANNOgesic is a highly useful tool for the identification of sRNAs in this system and that its combination with molecular techniques is a successful way to identify various BvgAS-dependent and Hfq-binding sRNAs. IMPORTANCE Noncoding small RNAs (sRNAs) are crucial for posttranscriptional regulation of gene expression in all organisms and are known to be involved in the regulation of bacterial virulence. We have investigated the presence of sRNAs in the obligate human pathogen B. pertussis, using transcriptome sequencing (RNA-seq) and the recently developed prokaryotic sRNA search program ANNOgesic. This analysis has identified 143 sRNA candidates (90 previously unreported). We have classified their dependence on the B. pertussis two-component system required for virulence, namely, BvgAS, based on their expression in the presence/absence of the phosphorylated response regulator BvgA, confirmed several by Northern analyses, and demonstrated that 3 bind directly to B. pertussis Hfq, the RNA chaperone involved in mediating sRNA effects. Our study demonstrates the utility of combining RNA-seq, ANNOgesic, and molecular techniques to identify various BvgAS-dependent and Hfq-binding sRNAs, which may unveil the roles of sRNAs in pertussis pathogenesis.https://journals.asm.org/doi/10.1128/Spectrum.00044-21small RNAHfqBvgAS regulonpertussisRNA-seqANNOgesic |
| spellingShingle | Kyung Moon Minji Sim Chin-Hsien Tai Kyungyoon Yoo Charlotte Merzbacher Sung-Huan Yu David D. Kim Jaehyun Lee Konrad U. Förstner Qing Chen Scott Stibitz Leslie G. Knipling Deborah M. Hinton Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic Microbiology Spectrum small RNA Hfq BvgAS regulon pertussis RNA-seq ANNOgesic |
| title | Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic |
| title_full | Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic |
| title_fullStr | Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic |
| title_full_unstemmed | Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic |
| title_short | Identification of BvgA-Dependent and BvgA-Independent Small RNAs (sRNAs) in Bordetella pertussis Using the Prokaryotic sRNA Prediction Toolkit ANNOgesic |
| title_sort | identification of bvga dependent and bvga independent small rnas srnas in bordetella pertussis using the prokaryotic srna prediction toolkit annogesic |
| topic | small RNA Hfq BvgAS regulon pertussis RNA-seq ANNOgesic |
| url | https://journals.asm.org/doi/10.1128/Spectrum.00044-21 |
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