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Distinct early cellular kinetics in participants protected against colonization upon Bordetella pertussis challenge.

ABSTRACT: BACKGROUNDTo date, only limited data are available on the mechanisms of protection against colonization with Bordetella pertussis in humans.METHODSIn this study, the cellular responses to B. pertussis challenge were monitored longitudinally using high-dimensional EuroFlow-based flow cytometry, allowing quantitative detection of more than 250 different immune cell subsets in the blood of 15 healthy donors.RESULTSParticipants who were protected against colonization showed different early cellular responses compared with colonized participants. Especially prominent for colonization-protected participants were the early expansion of CD36- nonclassical monocytes on day 1 (D1), natural killer cells (D3), follicular T helper cells (D1-D3), and plasma cells (D3). Plasma cell expansion on D3 correlated negatively with the CFU load on D7 and D9 after challenge. Increased plasma cell maturation on D11-D14 was found in participants with seroconversion.CONCLUSIONThese early cellular immune responses following experimental infection can now be further characterized and potentially linked to an efficient mucosal immune response, preventing colonization. Ultimately, their presence may be used to evaluate whether new B. pertussis vaccine candidates are protective against B. pertussis colonization, e.g., by bacterial challenge after vaccination.TRIAL REGISTRATIONClinicalTrials.gov NCT03751514.FUNDINGInnovative Medicines Initiative 2 Joint Undertaking and the EuroFlow Consortium.

SUBMITTER: Diks AM

PROVIDER: S-EPMC9974097 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Distinct early cellular kinetics in participants protected against colonization upon Bordetella pertussis challenge.

Diks Annieck M AM de Graaf Hans H Teodosio Cristina C Groenland Rick J RJ de Mooij Bas B Ibrahim Muktar M Hill Alison R AR Read Robert C RC van Dongen Jacques Jm JJ Berkowska Magdalena A MA

The Journal of clinical investigation 20230301 5

BACKGROUNDTo date, only limited data are available on the mechanisms of protection against colonization with Bordetella pertussis in humans.METHODSIn this study, the cellular responses to B. pertussis challenge were monitored longitudinally using high-dimensional EuroFlow-based flow cytometry, allowing quantitative detection of more than 250 different immune cell subsets in the blood of 15 healthy donors.RESULTSParticipants who were protected against colonization showed different early cellular ...[more]

PMID: 36649086

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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.

Dataset Information

Distinct early cellular kinetics in participants protected against colonization upon Bordetella pertussis challenge.

Publications

Distinct early cellular kinetics in participants protected against colonization upon Bordetella pertussis challenge.

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