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Effects of guanidinium hydrochloride on the structure and immunological properties of Bordetella pertussis fimbriae.

ABSTRACT: Denaturation of Bordetella pertussis fimbrial preparations by guanidinium hydrochloride (GdnHCl) has been characterized using static light scattering, c.d., fluorescence and antibody recognition. The susceptibility of Fim2 + 3 (a mixed preparation of two fimbrial types) to GdnHCl was found to be highly dependent on pH; as the pH was increased from pH 7.2 to 10.5, the concentration of GdnHCl required to induce 50% denaturation was decreased. At pH 10.5, Fim2 + 3 was denatured by GdnHCl in a three-step pathway comprising: (1) formation of a pre-denaturational intermediate at less than 1.0 M-GdnHCl; (2) dissociation of the fimbrial polymer into subunits between 2 M- and 3.2 M-GdnHCl; and (3) subunit unfolding between 2.8 M- and 3.6 M-GdnHCl. A similar pathway was also found for the denaturation of the individual fimbrial types, Fim2 and Fim3, except that unfolding of either subunit commenced at a lower GdnHCl concentration (2.2 M) than that found for the mixture of fimbriae, Fim2 + 3. The second step in the denaturation pathway, dissociation into subunits, was partially reversible, but the renaturation and reassociation of fully unfolded subunits to form fimbriae-like structures was not achieved. These findings demonstrate that the GdnHCl denaturation of complex polymeric proteins is unlikely to follow a reversible two-state denaturation pathway, and support the involvement of a chaperone-like protein in the folding and assembly of the fimbriae in vivo. Measurement of the ability of anti-fimbrial monoclonal antibodies to recognize intermediates in the denaturation pathway enabled the identification of two types of epitope which were dependent on different aspects of fimbrial tertiary/quaternary structure.

SUBMITTER: Pearce AM

PROVIDER: S-EPMC1130960 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

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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.

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Effects of guanidinium hydrochloride on the structure and immunological properties of Bordetella pertussis fimbriae.

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