Pertussis Toxin Operon Research Articles

A controlled study of the relationship between Bordetella pertussis infections and sudden unexpected deaths among German infants.

A controlled study of the relationship between Bordetella pertussis infections and sudden unexpected deaths among German infants.

Construction and characterisation of Salmonella typhimurium aroA simultaneously expressing the five pertussis toxin subunits

Pertussis toxin (PT) is a major protective antigen of Bordetella pertussis, the causative agent of whooping cough. Current vaccines against whooping cough are administered parenterally, and generate a systemic immune response. An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunisation with live vaccine strains of Salmonella spp. We have individually expressed the five PT subunits in Salmonella typhimurium aroA and subsequently expressed a synthetic pertussis toxin operon (pDP16), via the use of a temperature inducible expression system. In S. typhimurium aroA containing individual subunit expression plasmids the S5 subunit was found to be completely processed, while all other subunits were partially processed. In S. typhimurium aroA (pDP16) S5 was completely processed, S1 was partially processed, whilst S2, S3 and S4 remained unprocessed. Induction of the synthetic PT operon decreased the in vitro invasiveness of S. typhimurium aroA, compared to vector-only and plasmid-less strains. Following oral immunisation of Balb c mice with S. typhimurium aroA (pDP16), statistically significant IgG ( p < 0.05) specific to PT was detected in the serum of mice. Nonetheless, despite a statistically significant anti-PT serum antibody response in immunised mice, vaccination with S. typhimurium aroA (pDP16) failed to protect mice from virulent challenge.

Expression of a synthetic pertussis toxin operon in Escherichia coli

Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated bouts of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A B bacterial toxin consisting of five subunits S1–S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized vnto an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli β-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

The pertussis toxin liberation genes of Bordetella pertussis are transcriptionally linked to the pertussis toxin operon.

The DNA sequence of the pertussis toxin operon (ptx) of Bordetella pertussis predicts that transcription of the operon ends downstream from the ptxS3 gene at a possible stem-loop structure. Secretion of the assembled pertussis toxin into the culture medium required the expression of 8 genes arranged in an operon (ptl) and lying 55 bp downstream from the ptx and ptl operons are cotranscribed and coregulated by the P(TOX) promoter. Deletion of the 55-bp DNA region caused an increase in the amount of the ptl transcripts. It is likely that this DNA region is involved in regulation of the ptx-pti expression.

Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis.

Regulation of virulence factor expression in Bordetella pertussis is mediated by the BvgAS two-component regulatory system. Although previous studies have demonstrated that the transcriptional regulation of the filamentous hemagglutinin gene (fhaB) involves binding of the BvgA activator directly to the fhaB promoter region, the mechanism of pertussis toxin operon (ptx) regulation by BvgA has remained unclear. We demonstrate in vitro the specific binding of BvgA to a region upstream of the ptx promoter that encompasses a 20-bp directly repeated sequence (positions -157 to -117) previously shown to be critical for BvgA-dependent activation. This binding is strictly dependent on the phosphorylation of BvgA, which can be obtained by incubation of BvgA with acetyl phosphate. By DNase I protection studies, we demonstrate the synergistic binding of BvgA-phosphate and purified Escherichia coli RNA polymerase to the ptx promoter. In the presence of the polymerase holoenzyme, a greatly extended footprint encompassing the region between -163 and the putative polymerase binding site was observed. The implications of these observations for pertussis toxin expression and regulation are discussed.

Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli.

Transcription of the pertussis toxin operon (ptx) is positively regulated in Bordetella pertussis by the bvgAS locus. However, a ptx-lacZ transcriptional fusion in Escherichia coli cannot be activated by bvgAS in trans. This suggests that an additional factor(s) is required for transcription of ptx. A gene encoding a Bvg accessory factor (Baf) was identified by its ability to activate an E. coli ptx-lacZ fusion in the presence of bvgAS. The expression of ptx-lacZ was decreased by the addition of 40 mM MgSO4, a compound that also modulates ptx expression in B. pertussis. Baf alone did not activate expression of an E. coli fhaB-lacZ fusion, nor did it increase expression of fhaB-lacZ in trans with bvgAS. The gene encoding Baf was localized, sequenced, and found to produce a novel 28-kDa protein. Sequences homologous to B. pertussis baf were identified in Bordetella bronchiseptica and Bordetella parapertussis but not in Bordetella avium. When an additional copy of baf was integrated into the chromosome of BC75, a B. pertussis mutant that produces a low level of pertussis toxin, pertussis toxin production was partially complemented in the cointegrate strain.

Polymerase chain reaction identification of Bordetella pertussis infections in vaccinees and family members in a pertussis vaccine efficacy trial in Germany

The polymerase chain reaction (PCR) was recently added to conventional culture and serology for the diagnoses of Bordetella pertussis infection in a large vaccine efficacy trial in Germany. In vaccinees or family members who had illnesses with cough, two nasopharyngeal swabs (calcium alginate for culture and Dacron for PCR) were taken and initial and follow-up clinical data were obtained. PCR was done using oligonucleotide primers PTp1 and PTp2 which amplify a 191-base pair DNA fragment of pertussis toxin operon. From December, 1993, to May, 1994, 555 pairs of swabs were processed; 28 grew B. pertussis and 9 grew B. parapertussis. Twenty-six of the 28 subjects with B. pertussis-positive cultures also had positive PCR results as did one of the 9 B. parapertussis cases and 82 additional samples were positive by PCR. PCR increased the identification of subjects with B. pertussis infections by almost 4-fold. Clinical characteristics were analyzed by laboratory category (Group 1, 28 culture-positive; Group 2, 82 culture-negative, PCR-positive; and Group 3, 436 culture- and PCR-negative). Group 1 subjects were more likely to have a diagnosis of definite or probable pertussis and to have paroxysmal cough, posttussive vomiting, whooping and a cough duration of > or = 4 weeks than Group 2 or 3 subjects. In contrast Group 2 subjects were more likely than Group 1 subjects to have had previous pertussis immunization or prior antibiotics. PCR identified many mild illnesses caused by B. pertussis that were not identified by culture.

The virB operon of the Agrobacterium tumefaciens virulence regulon has sequence similarities to B, C and D open reading frames downstream of the pertussis toxin-operon and to the DNA transfer-operons of broad-host-range conjugative plasmids.

The virB operon of the Agrobacterium tumefaciens virulence regulon has sequence similarities to B, C and D open reading frames downstream of the pertussis toxin-operon and to the DNA transfer-operons of broad-host-range conjugative plasmids.

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations.

Further analysis of the sequence of the S1 subunit of pertussis toxin

The two published sequences of the pertussis toxin operon differ in 3 bp in the S1 subunit gene. In this report, we provide evidence that Bordetella pertussis strains are able to produce active pertussis toxin only when they contain one of the two possible nucleotide sequences.

Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli

The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.

Identification of a DNA fragment in the genome of Bordetella pertussis carrying repeated DNA sequences also present in other Bordetella species

A DNA fragment (3.8 kbp) which hybridized to repeated sequences in the genome of Bordetella species has been cloned from Bordetella pertussis chromosomal DNA. Eleven subclones were constructed from this fragment. They exhibited distinct inter-species hybridization patterns in genomic blots of each of the Bordetellae used in the study. One subclone revealed intra-species variability among B. pertussis strains and another did not hybridize to B. parapertussis. The 3.8 kbp DNA fragment possesses a middle sequence surrounded by repeated sequences organized in an opposite symmetrical orientation. The external inverted repeats of it hybridized to a 680 bp DNA sequence which is located about 800 bp upstream of the pertussis toxin operon. The novel structural organization of the 3.8 kbp fragment suggests the possibility that this DNA segment is an IS-like element which may have an important function in the expression of virulence determinants in Bordetella bacteria.

Specific identification of Bordetella pertussis by the polymerase chain reaction

Oligonucleotide primers were used to amplify specific DNA regions of the Bordetella pertussis genome by the polymerase chain reaction. One pair of primers, PTp1/PTp2, identified a 191-bp DNA fragment located in the regulatory region of the pertussis toxin operon; a second pair of primers led to amplification of a 121-bp DNA piece located in an insertion-like element specific to B. pertussis. Both sets of primers were able to discriminate between the pathogen and related Bordetella species; they detected down to 6 bacteria and appeared suitable for routine detection of B. pertussis in clinical specimens.

Promoter of the pertussis toxin operon and production of pertussis toxin

Pertussis toxin (PT), the major virulence factor of Bordetella pertussis, is composed of five different subunits whose genes are organized as an operon. We report the mapping of the promoter region of the PT operon and show that this promoter is only weakly active in Escherichia coli. Bordetella parapertussis and Bordetella bronchiseptica, which do not produce any PT, are shown to have a weaker promoter sequence for this operon and not to produce any detectable PT mRNA. We show that transcription of the PT operon in B. pertussis was constant throughout until the late stationary phase, when transcription significantly decreased. Analysis of the transposon Tn5 mutant BP347 showed that the product of the vir locus was required for transcription of the PT operon. Characterization of the Tn5 mutant BP356 showed that subunit S3 was required for the release of PT into the extracellular medium.