Molecular Evolution of the Two-Component System BvgAS Involved in Virulence Regulation in Bordetella

The whooping cough agent Bordetella pertussis is closely related to Bordetella bronchiseptica, which is responsible for chronic respiratory infections in various mammals and is occasionally found in humans, and to Bordetella parapertussis, one lineage of which causes mild whooping cough in humans and the other ovine respiratory infections. All three species produce similar sets of virulence factors that are co-regulated by the two-component system BvgAS. We characterized the molecular diversity of BvgAS in Bordetella by sequencing the two genes from a large number of diverse isolates. The response regulator BvgA is virtually invariant, indicating strong functional constraints. In contrast, the multi-domain sensor kinase BvgS has evolved into two different types. The pertussis type is found in B. pertussis and in a lineage of essentially human-associated B. bronchiseptica, while the bronchiseptica type is associated with the majority of B. bronchiseptica and both ovine and human B. parapertussis. BvgS is monomorphic in B. pertussis, suggesting optimal adaptation or a recent population bottleneck. The degree of diversity of the bronchiseptica type BvgS is markedly different between domains, indicating distinct evolutionary pressures. Thus, absolute conservation of the putative solute-binding cavities of the two periplasmic Venus Fly Trap (VFT) domains suggests that common signals are perceived in all three species, while the external surfaces of these domains vary more extensively. Co-evolution of the surfaces of the two VFT domains in each type and domain swapping experiments indicate that signal transduction in the periplasmic region may be type-specific. The two distinct evolutionary solutions for BvgS confirm that B. pertussis has emerged from a specific B. bronchiseptica lineage. The invariant regions of BvgS point to essential parts for its molecular mechanism, while the variable regions may indicate adaptations to different lifestyles. The repertoire of BvgS sequences will pave the way for functional analyses of this prototypic system

Detection of small RNAs in Bordetella pertussis and identification of a novel repeated genetic element

Background: Small bacterial RNAs (sRNAs) have been shown to participate in the regulation of gene expression and have been identified in numerous prokaryotic species. Some of them are involved in the regulation of virulence in pathogenic bacteria. So far, little is known about sRNAs in Bordetella, and only very few sRNAs have been identified in the genome of Bordetella pertussis, the causative agent of whooping cough. Results: An in silico approach was used to predict sRNAs genes in intergenic regions of the B. pertussis genome. The genome sequences of B. pertussis, Bordetella parapertussis, Bordetella bronchiseptica and Bordetella avium were compared using a Blast, and significant hits were analyzed using RNAz. Twenty-three candidate regions were obtained, including regions encoding the already documented 6S RNA, and the GCVT and FMN riboswitches. The existence of sRNAs was verified by Northern blot analyses, and transcripts were detected for 13 out of the 20 additional candidates. These new sRNAs were named Bordetella pertussis RNAs, bpr. The expression of 4 of them differed between the early, exponential and late growth phases, and one of them, bprJ2, was found to be under the control of BvgA/BvgS two-component regulatory system of Bordetella virulence. A phylogenetic study of the bprJ sequence revealed a novel, so far undocumented repeat of ~90 bp, found in numerous copies in the Bordetella genomes and in that of other Betaproteobacteria. This repeat exhibits certain features of mobil

Sequential activation and environmental regulation of virulence genes in Bordetella pertussis.

Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level

Characterization of a hepatitis A virus strain suitable for vaccine production

A novel isolate of hepatitis A virus, obtained from a clinical sample, has been adapted to grow on cultured human diploid cells. Growth and purification parameters have been optimized to obtain conditions suitable for the development of an inactivated vaccine. The entire viral genome was molecularly cloned, and the gene encoding the VP3 capsid protein was expressed in Escherichia coli. The resulting recombinant VP3 was used to obtain rabbit antisera which recognize the denatured protein in purified virion preparations. Nucleotide sequencing data are presented and compared to known sequences of different strains

Response of the bvg regulon of Bordetella pertussis to different temperatures and short-term temperature shifts

Bordetella pertussis produces a number of virulence factors whose expression is coordinately regulated by the bvgAS locus. Transcription of virulence genes is repressed by environmental factors such as low temperature (25°C) and chemical stimuli. Temperature shift of bacterial cultures from 25°C to 37°C activates two classes of bvg-regulated virulence genes: the early genes, which are activated within 10 min, and late genes, which require 2-4 h for activation. During the interval between the activation of the early and late genes, the intracellular concentration of BvgA increases 50-fold. It has been proposed that this increased concentration may be required for the activation of the late genes. Here we have analysed the response of the bvg locus to intermediate temperatures and to repeated temperature shifts. Temperature shifts of B. pertussis cultures from 22°C to 28°C, 32°C or 35°C resulted in the synthesis of low, intermediate, and high amounts of BvgA. This implied that the intracellular concentration of BvgA is temperature-dependent. We have also observed that the amount of virulence factors produced correlates with the BvgA concentration. When bacteria grown at 37°C were shifted to 22°C, transcription from the adenylate cyclase toxin haemolysis promoter (P(AC)) was repressed after 30 min, while transcription from the bvg (P1,) and filamentous haemagglutinin (P(FHA)) promoters was repressed after 2 h. During this time, the amount of BvgA did not decrease. A subsequent temperature shift from 22°C to 37°C induced transcription from the P1 and P(FHA) promoters after 10 min and transcription from the P(AC) promoter after 20 min. This result shows that in the presence of a high concentration of BvgA, the time lag between temperature shift and late promoter transcription is reduced from 2-4 h to 20 min. The above data support the proposal that the concentration of BvgA plays a role in activating expression of the late genes

Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis

none4noRegulation of the genes coding for virulence factors in Bordetella pertussis is controlled by the bvg locus, which encodes one putative sensory protein (BvgS) and one positive regulator of transcription (BvgA). We have studied the transcription of the bvg locus and found that this is controlled by a 350-base-pair DNA fragment, which contains five promoters, three of which transcribe the bvg locus, one transcribes an antisense RNA, and one transcribes a virulence-associated gene. Under noninducing conditions, only the promoter P2 is active and this is responsible for the production of low amounts of regulatory proteins. Upon induction, the other four promoters become active and, by a mechanism that may involve transcriptional and translational regulation, cause a 50-fold increase of the transcriptional activator BvgA. A model of the autoregulation of the bvg locus is presented.noneScarlato V.; Prugnola A.; Arico B.; Rappuoli R.Scarlato V.; Prugnola A.; Arico B.; Rappuoli R