Large scale cultivation of Bordetella pertussis in submerged culture for production of pertussis toxin

Summary Large quantities of purified Bordetella pertussis toxin were required to develop an investigational toxoid vaccine. To overcome the difficulties associated with the large scale growth of Bordetella pertussis in submerged culture, we employed an aeration strategy using compressed oxygen and an antifoam agent. The microorganism grown in this way in 1001 batches for 20–24 h yielded 4–6 mg·1^-1 pertussis toxin.     

The effect of methylated cyclodextrin on pertussis toxin accumulation in a Bordetella pertussis culture in a bioreactor

The results obtained in the study of the influence methylated cyclodextrin (beta CD) on the growth of B. pertussis and the accumulation of pertussis toxin in the course of submerged batch cultivation in a bioreactor are presented. As demonstrated by these results, the presence of beta CD in the culture medium in a dose of 0.1 ml/l in the growth deceleration phase causes a tenfold increase in the synthesis of pertussis toxin by microbial cells in comparison with conditions characterized by the absence of beta CD. It cannot by ruled out that beta CD may act as a stressor which influences the synthesis of pertussis toxin, protector protein making it possible for the microbe to survive under new conditions.     

Trial of pertussis toxin activity in vitro on CHO cells

The activity of B. pertussis toxin has been tested in the continuous culture of CHO (Chinese hamster ovary) cells. The in vitro method of testing B. pertussis toxin is rapid, highly sensitive and specific. The unit of activity of B. pertussis toxin is higher than in mouse tests by several orders. The specificity of the action of B. pertussis toxin on CHO cells has been confirmed by the test of the neutralization of the toxicity effect with antiserum.     

Different concentrations of pertussis toxin have opposite effects on agonist-induced PGE2 formation in mesangial cells

Pertussis toxin may inactivate N proteins linked to phospholipase C. We examined the effect of pretreatment with pertussis toxin at different concentrations and times on agonist-induced PGE2 synthesis in mesangial cells. Two to four hours with 10-50 ng/ml of pertussis toxin inhibited the response to angiotensin and platelet activating factor, but with a different sensitivity. This was associated with decreased [14C]arachidonic acid release in prelabeled cells. The response to A23187 was unaltered. At high concentrations (1 to 5 micrograms/ml) pertussis toxin increased basal PGE2 and the response to all agonists. Pertussis toxin pretreatment resulted in a dose-dependent ribosylation of a 40 kDa protein band. Thus, responses to different agonists have different sensitivity to pertussis toxin inhibition, which at high concentrations may even have opposite effects.     

Differential effects of cholera toxin and pertussis toxin on the c-fos and c-jun mRNA expression in rat C6 glioma cells

Cholera toxin (CTX) increased c-fos mRNA level whereas it down-regulated the c-jun mRNA level in rat C6 glioma cells. In contrast to the action of CTX, pertussis toxin (PTX) did not affect either c-fos or c-jun mRNA level. The elevated c-fos mRNA level induced by CTX was significantly inhibited by the co-treatment with dexamethasone (DEX). However, DEX did not affect CTX-induced down-regulation of c-jun mRNA level. Cycloheximide (CHX) increased c-fos and c-jun mRNA levels. CHX caused a super-induction of CTX-induced c-fos mRNA level. Our results suggest that CTX-, but not PTX-, sensitive G-proteins may play an important role for c-fos mRNA up-regulation and c-jun mRNA down-regulation. In addition, DEX appears to have a selective inhibitory action against c-fos mRNA expression regulated by CTX. Ongoing protein synthesis inhibition is required for the superinduction of c-fos, but not c-jun, mRNA induced by CTX.     

Inhibitory effects of pertussis toxin on a depolarization-evoked Ca2+ influx in NG108-15 cells

Depolarized stimulation 1.5-fold increased Ca2+ influx which was inhibited by pretreatment with verapamil or LaCl3. Treatment with pertussis toxin, islet-activating protein (IAP), induced a reduction in 50 mM K+-induced Ca2+ influx and stimulated adenylate cyclase (AC) activity in NG108-15 cells. However, addition of dibutyryl cAMP or forskolin treatment elevating cAMP level exerted no effects on a depolarization-induced Ca2+ influx. Dissociated B-oligomer of IAP after treatment with dithiothreitol and ATP increased a depolarization-evoked Ca2+ influx. It is suggested that inhibitory GTP-binding protein (G1) or other IAP substrate proteins could directly be involved in Ca2+ influx via voltage-sensitive Ca2+ channel.     

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.     

Dissociating the enhancing and inhibitory effects of pertussis toxin on autoimmune disease

Pertussis toxin (PT) has both enhancing and inhibitory effects on experimental autoimmune disease, depending on its time of administration relative to immunization. The inhibitory effect is due to blocking of G(i)-coupled receptors by the enzymatic A subunit. In this study, we attribute the enhancing effect of PT to the cell-binding B subunit (PT-B). C57BL/6 mice, a strain that requires PT to develop experimental uveitis, were immunized with a retinal Ag and were injected with whole PT, PT-B, or vehicle. Disease and associated immunological responses were evaluated. The results showed that PT-B, determined to be free of biologically significant contamination with whole PT or with endotoxin, was able to mimic all the effects of PT with respect to disease induction, enhancement of delayed-type hypersensitivity, enhancement of lymphocyte proliferation, induction of an innate IL-12 response, and promotion of an adaptive IFN-gamma response to the uveitogenic Ag. Our results suggest that PT-B is largely responsible for the disease-enhancing properties of PT.     

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin

Together with macrophages and dendritic cells, mast cells have recently been shown to interact with certain pathogenic bacteria and present microbial antigens to the immune system. We show here that Bordetella pertussis can adhere to and be phagocytosed by mast cells. In addition, mast cells are able to process and present B. pertussis antigens to T lymphocytes. Furthermore, exposure of mast cells to B. pertussis induced the release of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). The release of IL-6 was strongly reduced by pertussis toxin expressed by B. pertussis . The production of IL-10, but not that of IL-4, by mast cells was also inhibited by pertussis toxin. Depletion of mast cells in vivo resulted in significant reduction of early TNF-α production in bronchoalveolar lavage (BAL) fluids of B. pertussis -infected mice. These data suggest that mast cells may play a role in the induction of immune responses against B. pertussis through the release of cytokines, especially TNF-α.     

Interaction of pertussis toxin with cells and model membranes.

The interaction of pertussis toxin (PT) with cells and model membranes was investigated by examining PT-induced intoxication of Chinese hamster ovary cells and by studying the binding of PT and its subunits to phospholipid vesicles. Since certain bacterial toxins require an acidic environment for efficient interaction with membranes and subsequent entry into the cell, the requirement for an acidic environment for PT action was examined. PT, unlike bacterial toxins such as diphtheria toxin, did not require an acidic environment for efficient intoxication of Chinese hamster ovary cells. Potential modes by which PT might interact with biological membranes were studied by examining the binding of PT to a model membrane system. PT was found to be capable of interacting with phospholipid vesicles, however, efficient binding of the toxin to the vesicles occurred only in the presence of both ATP and reducing agent. The A subunit portion of the toxin bound preferentially to the vesicles while little binding of the B oligomer portion of PT to the model membranes was observed. Isolated A subunit, in the absence of the B oligomer, also bound to the vesicles with optimal binding occurring in the presence of reducing agent. After cleavage of the A subunit by trypsin, probably at Arg-181, Arg-182, and/or Arg-193, large fragments which lacked the C-terminal portion of the A subunit of PT no longer associated with the lipid vesicles. These results suggest that the A subunit of PT can interact directly with a lipid matrix and, if freed from the constraints imposed by the B oligomer, may be capable of interacting with cellular membranes.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

Enhancement of differentiation of rat adipocyte precursor cells by pertussis toxin

To examine whether GTP-binding protein(s) is (are) involved in adipocyte differentiation, the effect of pertussis toxin (PT) was studied in rat adipocyte precursor cell culture. PT potentiated adipose conversion induced by dexamethasone, insulin, and 1-methyl-3-isobutylxanthine in a dose- and time-dependent fashion. Attenuation of an inhibitory control of adenylate cyclase was not the mechanism of action of PT. The dose-dependent inhibition of PT-catalyzed ADP-ribosylation of the Mr 40,000 protein of the cell membrane by preincubation of the toxin was inversely related to the potentiating effect on differentiation. PT-sensitive G protein(s) may be involved in adipocyte differentiation in a negative fashion.     

Adhesion of lymphoid cells to fibroblasts in tissue culture

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.     

Platelet-activating factor mediated effects on human neutrophil function are inhibited by pertussis toxin

Pretreatment of human neutrophils with pertussis toxin inhibits platelet-activating factor-mediated chemotaxis, superoxide generation, aggregation, and release of lysozyme. By contrast, superoxide generation observed in the presence of phorbol-12-myristate-13 acetate is unaffected. Our results suggest that a target protein for pertussis toxin, probably a GTP binding protein termed "Ni", is involved in the actions of platelet-activating factor on human neutrophils.