Dataset Information

Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment.

ABSTRACT: Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the BvgAS two-component system, a master regulator of virulence. Our results show that the expression of this regulatory circuit is downregulated upon internalization. Moreover, we demonstrate that the switch to the avirulent Bvg^- phase augments a very complex process based on the adjustment of central and energy metabolism, cell wall reinforcement, maintenance of appropriate redox and metal homeostasis, and repair of damaged macromolecules. Nevertheless, not all observed effects could be simply attributed to the transition to Bvg^- phase, suggesting that additional regulators are involved in the adaptation to the intramacrophage environment. Interestingly, a large number of genes required for the metabolism of sulphur were strongly modulated within macrophages. In particular, the mutant lacking two genes encoding cysteine dioxygenases displayed strongly attenuated cytotoxicity toward THP-1 cells. Collectively, our results suggest that intracellular B. pertussis cells have adopted the Bvg^- mode to acclimate to the intramacrophage environment and respond to antimicrobial activities elicited by THP-1 cells. Therefore, we hypothesize that the avirulent phase represents an authentic phenotype of internalized B. pertussis cells.

SUBMITTER: Farman MR

PROVIDER: S-EPMC9858536 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment.

Farman Mariam R MR Petráčková Denisa D Kumar Dilip D Držmíšek Jakub J Saha Argha A Čurnová Ivana I Čapek Jan J Hejnarová Václava V Amman Fabian F Hofacker Ivo I Večerek Branislav B

Emerging microbes & infections 20231201 1

Bordetella pertussis, the causative agent of whooping cough, is an extracellular, strictly human pathogen. However, it has been shown that B. pertussis cells can escape phagocytic killing and survive in macrophages upon internalization. Our time-resolved RNA-seq data suggest that B. pertussis efficiently adapts to the intramacrophage environment and responds to host bactericidal activities. We show that this adaptive response is multifaceted and, surprisingly, related to the ...[more]

PMID: 36357372

Similar Datasets

Project description:Purpose: Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due at least in part to the introduction of a less effective acellular vaccine in the 1990’s. It is crucial then to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA/BvgS is the B. pertussis master regulator that regulates the expression of various virulence genes. Previous genome wide studies to identify the bvgAS regulon in B. pertussis have been limited to microarray analyses. The goal of this study is to define the bvgAS regulon using NGS based RNA-seq analysis and RT-qPCR and to compare the differences with the previous microarray data. Method: To define the bvgAS regulon, transcriptomes were generated using B. pertussis Tohama I BP536 (WT) and a ΔbvgAS derivative in the presence and absence of phosphorylated BvgA. Bacteria were grown either under modulating (50 mM MgSO4) conditions, in which phosphorylated BvgA is undetectable (Bvg- mode), or non-modulating (no MgSO4) conditions in which BvgA is phosphorylated (Bvg+ mode). After total RNA extraction and rRNA removal, strand-specific DNA libraries were prepared for Illumina sequencing using the ScriptSeq 2.0 kit (Illumina) in duplicates. Libraries were sequenced using a HiSeq 2000 sequencer (Illumina; University of Buffalo Next Generation Sequencing Core Facility). Sequence reads were mapped to the reference genome (NC_002929.2) and normalized against total reads. Differential expression analyses were performed using CLC Biomedical Genomic Workbench version 3.5.4 with default parameters. Differential gene expression profiles were obtained after Empirical DGE statistical test with FDR p-value. RT–qPCR validation was performed using SYBR Green assays. Result: Using CLC Biomedical Genomic Workbench version 3.5.4, ~ 20-25 million reads per sample were mapped to the reference B. pertussis genome (NC_002929.2). RNA-seq analyses identified >550 genes (15% of genome) that were regulated in a bvgAS-dependent manner with a fold difference ≥ 1.6 and FDR p-value < 0.05. RT-qPCR was used for confirmatory analyses for 80 genes. Overall, we identified 245 genes that were positively regulated and 326 genes that were negatively regulated by bvgAS. 362 members of the bvgAS regulon were newly identified by our study. Most importantly, our analyses indicated that the expression of dozens of transcriptional regulators increases, while the expression of multiple metabolic genes decreases in the presence of BvgA~P. Conclusion: We report here the first RNA-seq analysis of the bvgAS regulon in B. pertussis, revealing that > 550 genes are regulated by bvgAS. Our results suggest that metabolic changes in the Bvg- mode (absence of BvgA phosphorylation) may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new genes negatively regulated by bvgAS that can be tested for function.

Project description: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 (BvgA~P), and virulence-activated genes (vags) are expressed [Bvg(+) mode]. When BvgS is inactive and BvgA is not phosphorylated, virulence-repressed genes (vrgs) are induced [Bvg(-) mode]. Here, we have 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 reveal more than 550 BvgA-regulated genes, of which 353 are 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 BvgA~P as an apex regulator of transcriptional networks promoting virulence. Using in vitro transcription, we demonstrate that the promoter for brpL is directly activated by BvgA~P. BvgA-FeBABE cleavage reactions identify BvgA~P binding sites centered at positions -41.5 and -63.5 in bprL Most importantly, we show 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 suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new vrgs that can be tested for function.IMPORTANCE Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due, at least in part, to the introduction of a less effective acellular vaccine in the 1990s. It is crucial, then, to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA (response regulator)/BvgS (histidine kinase) is the master regulator of B. pertussis virulence genes. We report here the first RNA-seq analysis of the BvgAS regulon in B. pertussis, revealing that more than 550 genes are regulated by BvgAS. We show that genes for multiple and varied metabolic pathways are highly regulated in the Bvg(-) mode (absence of BvgA phosphorylation). Our results suggest that metabolic changes in the Bvg(-) mode may be participating in bacterial survival, transmission, and/or persistence.

Dataset Information

Avirulent phenotype promotes Bordetella pertussis adaptation to the intramacrophage environment.

Publications

Avirulent phenotype promotes <i>Bordetella pertussis</i> adaptation to the intramacrophage environment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets