Genetics of pertussis toxin

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis. The cloning and nucleotide sequencing of the PT genes from B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica has elucidated the evolution of the Bordetella species and allowed considerable advances towards the understanding of their gene expression and the development of safer vaccines against pertussis.     

Use of cyclodextrin as an agent to induce excretion of Bordetella pertussis antigens

Abstract This paper attempts to provide an explanation for the effect of cyclodextrin on the yield of Bordetella pertussis soluble antigens. It was demonstrated that the addition of cyclodextrin to the synthetic Stainer-Scholte liquid medium enhances the level of the intracellular form of adenylate cyclase (200 kDa) in the supernate. In addition to this effect, it has been reported that cyclodextrin also enhances the levels of two other extracellular proteins, pertussis toxin and filamentous hemagglutinin. As these antigens are structurally different, it seems that the effect of cyclodextrin is not specific. With the use of different buffer systems of well-known action on outer membrane stability it was possible to determine a relationship between the presence of cyclodextrin, destabilisation of the outer membrane and the release of proteins. It was determined that the cyclodextrin did not modify the fluidity of B. pertussis cells but produced a change of outer membrane permeability.     

A simple novel approach for the purification of pertussis toxin

Abstract Pertussis toxin (PT) was purified from concentrated culture supernatants of Bordetella pertussis by a single step based on affinity chromatography on heparin-Sepharose 6B with a recovery of 36% in two fractions. The fraction of highest specific activity (0.91 mg of toxin per mg protein) constituted 15.3% of toxin content in crude material and appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibited the five bands corresponding to toxin subunits. The purified fraction induced clustering of Chinese hamster ovary cells at as little as 0.06 ng per ml. PT solution gave a single precipitation line with rabbit immune serum raised against crude Bordetella pertussis supernatant.     

Release of pertussis toxin and its interaction with outer-membrane antigens

The absence of subunit S3 in cell-associated pertussis toxin (PT) from a mutant of Bordetella pertussis which failed to produce cell-free toxin suggested that this subunit was involved in the release of PT into the culture medium. The addition of methylated beta-cyclodextrin (MCD) to the culture medium caused a small but consistent increase in the release of lipopolysaccharide (LPS) by four wild-type strains of B. pertussis. Since previous studies have shown that MCD also enhances the levels of PT in culture supernates, it seemed probable that the increased shedding of outer-membranes vesicles (OMV) may explain the increased levels of both cell-free PT and LPS. Release of PT was inhibited in media buffered with HEPES but was unaffected in Tris/HC1 buffer. This suggested that in addition to shedding of the outer membrane, increased permeability and greater destabilization of the outer membrane, as caused by Tris/HC1 buffer, may be important in the release of PT. Our data do not support the idea that PT is packaged into OMV because only an insignificant proportion (0.01%) of the total cell-free PT was associated with LPS. The association of PT with small micelles derived from outer-membrane amphiphiles may be more important since the LPS content of PT purified from culture supernates (containing no large OMV) was nearly 18% by weight.     

The effects of purified pertussis components and lipopolysaccharide on the results of the mouse weight gain test

The effects of highly purified components of Bordetella pertussis, that is pertussis toxin (PT) and filamentous haemagglutinin (FHA), and of lipopolysaccharide (LPS) were studied in the active mouse weight gain test (MWGT). The PT when given alone or with other components in various combinations caused weight losses and deaths 2-3 days after inoculation but FHA was not toxic in the MWGT. When FHA was given with PT, the toxic effect of PT was reduced. The LPS caused weight losses at 24 h which decreased when LPS was given with PT. The toxic effects of PT as indicated by late deaths and late weight losses or failure to gain weight continued until 14 days after inoculation. The various components had similar effects on mouse weight gain in both LACA and NIH strains of mice. The doses of PT used in the MWGT caused marked leucocytosis but FHA and LPS did not. No agglutinins appeared in the sera of mice inoculated with various purified components. The components were thus pure and did not contain agglutinogens.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.     

Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contributes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti-immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lymphocytes.     

Hemagglutination activities of purified pertussis toxin and filamentous hemagglutinin against erythrocytes from various animals

The hemagglutinating (HA) activities of purified pertussis toxin (PT) and filamentous hemagglutinin (FHA) were evaluated against unfixed and glutaraldehyde-fixed erythrocytes from ox, goose, horse, monkey, sheep, chicken, and rabbit. Both PT and FHA showed HA activities against fixed and unfixed erythrocytes from all the animals studied. The HA titers of FHA were higher than those of PT. The HA activities of FHA and PT were not destroyed completely even after heating these preparations at 56 C for 30 min. A simple test for the assay of PT in culture supernatants of Bordetella pertussis on the basis of HA activity has been described.     

A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) at doses as low as 0.8 ng.ml-1, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertussis, containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for pT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.     

Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis.

The ptl locus of Bordetella pertussis contains eight open reading frames which are predicted to encode proteins (PtlA to PtlH) that are essential for secretion of pertussis toxin from the bacterium and which are members of a family of transport proteins found in other types of bacteria. We have detected PtlE, PtlF, and PtlG in immunoblots of extracts of B. pertussis by using antibodies raised to fusion proteins consisting of maltose-binding protein and the individual Ptl proteins. These proteins have apparent molecular weights similar to those predicted by DNA sequence analysis. Cell fractionation studies indicated that all three Ptl proteins are associated with the membranes of B. pertussis, suggesting that the Ptl proteins form a gate or channel which facilitates transport of pertussis toxin. Cell extracts of other Bordetella spp. were probed with antibodies to Ptl proteins for the presence of these transport proteins. Neither Bordetella parapertussis nor Bordetella bronchiseptica contained detectable levels of PtlE or PtlF. This lack of detectable Ptl protein may provide an explanation for previous observations which indicated that introduction of the genes encoding pertussis toxin subunits from B. pertussis into other Bordetella spp. results in production of the toxin but not secretion of the toxin.     

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.     

Human cellular immune responses to Bordetella pertussis infection

We have compared the responses of peripheral blood leucocytes from three groups (i) patients suffering from pertussis (whooping cough), (ii) clinical staff caring for those patients and laboratory staff working with Bordetella pertussis, and (iii) staff with no known recent contact with B. pertussis. In vitro stimulation with filamentous haemagglutinin (FHA) caused significant increases in proliferation of only the patient group's lymphocytes. In vitro stimulation with pertussis toxin (PT) caused a large increase in proliferation of lymphocytes from all three groups and in the patient group the increase in proliferation was related to the dose of PT. Interleukin 2 (IL-2) production by leucocytes from all three groups was significantly increased following challenge with FHA or PT. The increases in IL-2 production were greatest in lymphocytes from patients with pertussis. Challenge with toxoided pertussis toxin had no effect on either proliferation or IL-2 production in any of the groups.     

Human cellular immune responses to Bordetella pertussis infection

Abstract We have compared the responses of peripheral blood leucocytes from three groups (i) patients suffering from pertussis (whooping cough), (ii) clinical staff caring for those patients and laboratory staff working with Bordetella pertussis , and (iii) staff with no known recent contact with B. pertussis . In vitro stimulation with filamentous haemagglutinin (FHA) caused significant increases in proliferation of only the patient group's lymphocytes. In vitro stimulation with pertussis toxin (PT) caused a large increase in proliferation of lymphocytes from all three groups and in the patient group the increase in proliferation was related to the dose of PT. Interleukin 2 (IL-2) production by leucocytes from all three groups was significantly increased following challenge with FHA or PT. The increases in IL-2 production were greatest in lymphocytes from patients with pertussis. Challenge with toxoided pertussis toxin had no effect on either proliferation or IL-2 production in any of the groups.     

Essential role of the consensus nucleotide-binding site of PtlH in secretion of pertussis toxin from Bordetella pertussis.

PtlH is a member of a specialized set of transport proteins that is essential for secretion of pertussis toxin (PT) from Bordetella pertussis. Previously, PtlH was shown to contain a consensus nucleotide-binding motif. Here, we demonstrate that introduction of plasmids containing mutant forms of ptlH, altered in the putative nucleotide-binding region, into a wild-type strain of B. pertussis resulted in inhibition of PT secretion. Thus, this region of PtlH appears to be essential for protein function. Moreover, the observed dominant negative phenotype suggests that PtlH either functions as a multimer or interacts with another component necessary for secretion of PT.     

Enhancement of Bordetella parapertussis infection by Bordetella pertussis in mixed infection of the respiratory tract

The epidemiological and pathogenic relationship between Bordetella pertussis and Bordetella parapertussis, the two causes of whooping cough (pertussis), is unclear. We hypothesized that B. pertussis, due to its immunosuppressive activities, might enhance B. parapertussis infection when the two species were present in a coinfection of the respiratory tract. The dynamics of this relationship were examined using the mouse intranasal inoculation model. Infection of the mouse respiratory tract by B. parapertussis was not only enhanced by the presence of B. pertussis, but B. parapertussis significantly outcompeted B. pertussis in this model. Staggered inoculation of the two organisms revealed that the advantage for B. parapertussis is established at an early stage of infection. Coadministration of PT enhanced B. parapertussis single infection, but had no effect on mixed infections. Mixed infection with a PT-deficient B. pertussis strain did not enhance B. parapertussis infection. Interestingly, the depletion of airway macrophages reversed the competitive relationship between these two organisms, but the depletion of neutrophils had no effect on mixed infection or B. parapertussis infection. We conclude that B. pertussis, through the action of PT, can enhance a B. parapertussis infection, possibly by an inhibitory effect on innate immunity.     

Antigenic heterogeneity in subunit S1 of pertussis toxin

Culture supernates containing pertussis toxin (PT) from four strains of Bordetella pertussis were examined for both immunological reactivity and biological activity. PT from all four strains sensitized mice to histamine and toxin was detectable in supernates of all strains when examined by Western blotting with polyclonal antiserum to PT. In supernates of three of the four strains, PT was detectable by an enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody to subunit S1 of PT as the third antibody layer. However, supernates from one strain, 18323, failed to react in ELISA. Electroblots probed with the monoclonal antibody labelled subunit S1 of PT from all strains except that of strain 18323. PT of strain 18323, whilst retaining histamine-sensitizing activity, differed antigenically from that of other strains.     

Quantitation of pertussis toxin in an enzyme linked immunosorbent assay with improved specificity

The quantitation of pertussis toxin (PT) in two sandwich ELISAs was tested for specificity. The detection of the captured PT was obtained by using either polyspecific rabbit anti Bordetella pertussis serum (RaBp-ELISA) or a monoclonal anti-PT antibody (McaPT-ELISA). No major differences in the estimation of PT in highly purified preparations were noted using either ELISA variants. In contrast, the quantitation of PT in crude extracts of B. pertussis cultures by the RaBp-ELISA was found to be over-estimated and showed greater variability when compared to the McaPT-ELISA. Comparison of the distribution of PT in the eluate fractions following partial purification by hydroxylapatite chromatography revealed that the results of the McaPT-ELISA were more specific as judged by SDS-PAGE analysis.