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1.
Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form. 相似文献
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Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera 总被引:1,自引:0,他引:1
Juan L. Arciniega Erik L. Hewlett Kathryn M. Edwards Drusilla L. Burns 《FEMS immunology and medical microbiology》1993,6(4):325-330
Abstract To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis. Antibodies reactive with ACT were the most prevalent in neonatal sera. Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived. Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice. Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli α-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with α-hemolysin. 相似文献
4.
Maria Bianchi Rinu Sivarajan Thorsten Walles Stephan Hackenberg Maria Steinke 《Innate immunity》2021,27(1):89
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. 相似文献
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《Microbes and infection / Institut Pasteur》2014,16(1):51-60
Despite high vaccination coverage, pertussis remains an important respiratory infectious disease and the least-controlled vaccine-preventable infectious disease in children. Natural infection with Bordetella pertussis is known to induce strong and long-lasting immunity that wanes later than vaccine-mediated immunity. Therefore, a live attenuated B. pertussis vaccine, named BPZE1, has been developed and has recently completed a phase I clinical trial in adult human volunteers. In this study, we investigated the contribution of adenylate cyclase (CyaA) in BPZE1-mediated protection against pertussis. A CyaA-deficient BPZE1 mutant was thus constructed. Absence of CyaA did not compromise the adherence properties of the bacteria onto mammalian cells. However, the CyaA-deficient mutant displayed a slight impairment in the ability to survive within macrophages compared to the parental BPZE1 strain. In vivo, whereas the protective efficacy of the CyaA-deficient mutant was comparable to the parental strain at a vaccine dose of 5 × 105 colony forming units (CFU), it was significantly impaired at a vaccine dose of 5 × 103 CFU. This impairment correlated with impaired lung colonization ability, and impaired IFN-γ production in the animal immunized with the CyaA-deficient BPZE1 mutant while the pertussis-specific antibody profile and Th17 response were comparable to those observed in BPZE1-immunized mice. Our findings thus support a role of CyaA in BPZE1-mediated protection through induction of cellular mediated immunity. 相似文献
6.
Protective effects of in vivo‐expressed autotransporters against Bordetella pertussis infection
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Koichiro Suzuki Naoaki Shinzawa Keisuke Ishigaki Keiji Nakamura Hiroyuki Abe Aya Fukui‐Miyazaki Kazuyoshi Ikuta Yasuhiko Horiguchi 《Microbiology and immunology》2017,61(9):371-379
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Bie Shung Tsai Robert J. Lefkowitz 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,587(1):28-41
We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg2+. The basal and prostaglandin E1-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E1. An increase in the concentration of Mg2+ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca2+. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn2+ for Mg2+ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E1-stimulated activity to a stimulatory effect. At a lower concentration of Mn2+ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase. 相似文献
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A virulent clone of Bordetella pertussis, injected intranasally into C57B1/6 or Balb/c mice, induced a respiratory tract infection that mimicked the infectious process of whooping cough. The density of the inoculum influenced the kinetics of in vivo bacterial growth, as well as the associated leucocytosis, which were of equivalent intensity in both strains of mice. Convalescing mice became resistant to re-infection, but not to the effects of the leucocytosis-promoting factor of the pertussis toxin. Prominent immune response, associated with acquired resistance, was a delayed type hypersensitivity (DTH) reaction, the intensity of which depended upon the genotype of the mice when the eliciting antigen contained the pertussis toxin in a biologically active form. Intranasal infection of congenic mice may represent an improved quantitative test for reproducible measurement of virulence and immunogenicity of B. pertussis. 相似文献
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Rodriguez Maria E. Hozbor Daniela F. Samo Analia L. Ertola Rodolfo Yantorno Osvaldo M. 《Journal of industrial microbiology & biotechnology》1994,13(5):273-278
Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X
cell concentration (g L–1)
-
specific growth rate (h–1)
- m
maximum specific growth rate (h–1)
- D
dilution rate (h–1)
- S
concentration of growth rate-limiting nutrient (glutamate) (mmol L–1 or g L–1)
- Ks
substrate saturation constant (mol L–1)
- ms
maintenance coefficient (g g–1 h–1)
- Yx/s
theoretical yield of cells from glutamate (g g–1)
- Yx/s
yield of cells from glutamate (g g–1)
- YPT/s
yield of soluble PT from glutamate (mg g–1)
- YKDO/s
yield of cell-free KDO from glutamate (g g–1)
- YPT/x
specific yield of soluble PT (mg g–1)
- YKDO/x
specific yield of cell-free KDO (g g–1)
- qPT
specific soluble PT production rate (mg g–1 h–1)
- qKDO
specific cell-free KDO production rate (g g–1 h–1) 相似文献
10.
Sylvie Lenin Jean Michel Alonso Colette Brezin Murielle Rocancourt Olivier Poupel 《FEMS microbiology letters》1986,37(1):89-94
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. 相似文献
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Carlos Angel Espinosa-Vinals Jiri Masin Jana Holubova Ondrej Stanek David Jurnecka Radim Osicka Peter Sebo Ladislav Bumba 《The Journal of biological chemistry》2021,297(1)
The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I–V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132–1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562–1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295–1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion. 相似文献
13.
Mirosŀaw Sopel 《Cell and tissue research》1995,279(2):441-444
Adenylate cyclase activity was localized in the lactating mouse mammary gland using an ultrastructural histochemical technique. Reaction product was deposited on the plasma membrane of the myoepithelial cells adjacent to the secretory epithelium. No reaction product was encountered on the secretory epithelium. These findings suggest that the presence of cAMP, previously biochemically documented in lactating mammary gland, is mainly connected with myoepithelial cellular activity. The asymmetrical distribution of adenylate cyclase activity suggests that cAMP is involved in the intercellular communication between the secretory and myoepithelial cells and that the secretory epithelium takes part in the regulation of the contraction of myoepithelial cells. 相似文献
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Peter ebo Zohra Moukrim Milo Kalhous Niels Schaft Gilles Dadaglio Valeria Sheshko Catherine Fayolle Claude Leclerc 《FEMS immunology and medical microbiology》1999,26(2):167-173
Bordetella pertussis adenylate cyclase toxin (ACT) is one of the few known protein toxins penetrating directly into the cytosol of target cells across their cytoplasmic membrane without the need for endocytosis. This capacity of ACT was recently exploited for in vivo delivery of single viral CD8(+) T-epitopes into MHC class I-presenting cells and induction of protective antiviral cytotoxic T-cell (CTL) responses. Here, we have explored the potential of the cell-invasive adenylate cyclase domain of the toxin to deliver larger antigens by evaluating the epitope-specific CTL responses induced by constructs bearing one to four copies of the CD8(+) T-epitope from the nucleoprotein of the lymphocytic choriomeningitis virus. The increase in the number of copies of the epitope was accompanied by a moderate decrease of the specific cell invasiveness of the ACT protein and did not lead to further enhancement of the level of induced epitope-specific CTL cells in mice, as compared to ACT with a single copy of the epitope. These results demonstrate the capacity of ACT to deliver larger heterologous antigens comprising several epitopes for antigenic presentation in vivo. 相似文献
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Michal Svoboda Monique Lambert Jean Christophe 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,675(1):46-61
(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTPγS as nucleotide activator, cholecystokinin as peptide hormone, and GDPβS and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (EA/ETOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant k+2, and a deactivation process (rate constant koff) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant k2) and/or the dissociation of the intact nucleotide (rate constant k?1), so that EA/ETOT = k+1/(k+1 + k2 + k?a). (2) The hormone CCK-8 increased the value of k+1 with GTP dose-dependently, from 0.2 to 10.9 min?1. The value of k?1 increased 0.01 to 0.3 min?1 but the value of k2 was unaltered at 7 min?1, so that EA/ETOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 μg/ml allowed also a large increase in EA/ETOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the koff value of the GTP-activated enzyme (from 7 min? to 0.5 min?1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of k+1 (from 0.2 to 0.8 min?1) and the occurrence of a significant 0.3 min?1 value for k?1. 相似文献
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Marianne Pilaski Klaus-Dieter Voigt Wolfgang Walter 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,582(3):380-389
The membrane-bound adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) of isolated rat adrenal cortex cells can be rendered soluble using 0.02 M Lubrol 12A9. The solubilized enzyme can be filtered through Millipore filters with pores 0.22 μm in diameter. Using gel filtration, on Sephadex G-200, adenylate cyclase activity was eluted with a distribution coefficient of 0.139, whereas on Sephadex G-100 the activity was eluted in the excluded volume. Half-maximum activation of the postulated guanyl nucleotide regulator site of adenylate was achieved with 5′-guanylyl-imidodiphosphate at a concentration of 1 · 10?6 M. In contrast, however, using intact isolated rat adrenal cortex cells the guanyl nucleotide regulator site could not be stimulated by 5′-guanylyl-imidodiphosphate. 相似文献
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We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg2+ (5.0–50 mM), while it activates the same enzyme in the presence of Ca2+ (0.1–1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+ and the inhibitory effect of RMI 12330 A in the presence of Ca2+. 相似文献
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M.S. Nijjar A.W.Y. Au K.C. Chaudhary 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,677(1):153-159
The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the Km and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs. 相似文献
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S van Heyningen 《FEBS letters》1983,164(1):132-134
The active A1 peptide of cholera toxin was linked by a disulphide bond to the lectin of Wisteria floribunda. The resulting conjugate activated the adenylate cyclase of intact U937 or K562 cells at the same concentrations as native toxin did, but to a greater extent. Activation was inhibited by N-acetyl-D-galactosamine or by antisera to the lectin or peptide. The characteristic lag phase between addition of toxin to cells and activation of cyclase was not found with the conjugate or with free A1 peptide. 相似文献