Previous Article | Next Article ![]()
Infection and Immunity, June 1999, p. 2763-2768, Vol. 67, No. 6
Department of Bacterial and Blood Products,
National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo
208-0011, Japan,1 and Unité de
Biochimie des Régulations Cellulaires, Institut Pasteur, 75724 Paris Cedex 15, France2
Received 21 December 1998/Returned for modification 5 February
1999/Accepted 16 March 1999
The effect of Bordetella pertussis adenylate cyclase
toxin (ACT) on platelet aggregation was investigated. This
cell-invasive adenylate cyclase completely suppressed ADP (10 µM)-induced aggregation of rabbit platelets at 3 µg/ml and strongly
suppressed thrombin (0.2 U/ml)-induced aggregation at 10 µg/ml. The
suppression was accompanied by marked increase in platelet
intracellular cyclic AMP (cAMP) content and was diminished by the
anti-ACT monoclonal antibody B7E11. A catalytically inactive point
mutant of ACT did not show the suppressive effect. Since an increase of
cAMP content is a known cause of platelet dysfunction, these results
indicate that the observed platelet inactivation was due to the
catalytic activity of ACT through increase of intracellular cAMP.
The adenylate cyclase toxin (ACT) of
Bordetella pertussis is a 1,706-amino acid (aa) RTX toxin
(5, 18) which enters mammalian cells and converts
intracellular ATP to cyclic AMP (cAMP) in an unregulated way due to its
high catalytic activity in the presence of calmodulin (7, 21, 24,
55). Since calmodulin is absent from bacteria, it is likely that
ACT functions exclusively in mammalian cells.
The adenylate cyclase catalytic activity of this toxin is located in
the N-terminal 400-aa domain which upon interaction with mammalian
cells is internalized into the target cells across the cytoplasmic
membrane in the presence of calcium (17, 18, 34, 43). The
invasive activity strictly depends on the integrity of the
1,300-residue-long C-terminal part of the molecule; even a small
deletion of 60 aa completely impairs the invasive activity (27,
45). This C-terminal domain is also endowed with hemolytic activity (1, 12, 22, 44, 45) and is structurally closely related to RTX toxins, including Since cAMP has been generally recognized to be an important
intracellular second messenger, numerous studies have been performed to
clarify the role of ACT in whooping cough disease. Mutant B. pertussis strains deficient in ACT synthesis are known to be
unable to colonize the surface of respiratory tract (19,
53). In addition, ACT has been shown to inhibit phagocytic
functions (7, 41) and to induce apoptosis in macrophages
(23, 30). However, the primary target of ACT during B. pertussis infection has not yet been clearly identified.
The platelets are one of the major blood components, and their
dysfunction results in hemorrhage, which is considered to be one of the
complications of whooping cough (52). cAMP, on the other
hand, is known to be a potent suppressor of platelet aggregation (25, 37, 46, 56). We demonstrate here that ACT suppresses platelet aggregation in vitro through increase of intracellular cAMP
due to its catalytic activity and that ACT induces prolongation of
bleeding time in vivo.
Preparation of toxins.
Recombinant ACT (3) and
its catalytically inactive derivative ACTK58Q (27) were
expressed in E. coli XL1-Blue harboring separately plasmids
pCACT3 (3) and pT7CT7ACT-K58Q (constructed by Peter
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Suppression of Platelet Aggregation by
Bordetella pertussis Adenylate Cyclase Toxin
and
![]()
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-hemolysin of Escherichia
coli (8). The hemolysin domain forms a hemolytic pore
on sheep and human erythrocytes (44, 45) and on artificial
lipid bilayer membranes (2, 51), suggesting that ACT has a
very broad target cell specificity. ACT can invade and can elevate
intracellular cAMP levels in a wide variety of cell types, including
isolated and established human leukocytes (7, 14, 16, 26,
41), mouse neuroblastoma N1E-115 (11) and adrenal
tumor Y-1 (16) cells, Chinese hamster ovary cells (16,
20, 38, 42), and baby hamster kidney cells (54), as
well as human and sheep erythrocytes (1, 13, 43, 45).
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
ebo and kind gifts from Agnes Ullmann [Institut Pasteur]). Recombinant toxins were extracted from ultrasound-disrupted cell debris, which contained 60 to 70% of total cellular adenylate cyclase
activity (45, 48), with 8 M urea-50 mM Tris-HCl-0.2 mM
CaCl2 (pH 7.5) and then purified to close to homogeneity by DEAE-Sepharose Fast Flow chromatography as described by Sakamoto et al.
(45) (Fig. 1). Protein content
was determined with a Pierce bicinchoninic acid (BCA) protein assay kit
(49). Sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) on an 8% gel was performed as described by
Laemmli (35), and proteins were visualized with Coomassie
brilliant blue.

View larger version (38K):
[in a new window]
FIG. 1.
ACT and ACTK58Q preparations used in this study. ACT and
ACTK58Q were prepared as described in Materials and Methods by
DEAE-Sepharose Fast Flow chromatography. The preparations (20 µg of
protein/lane) were loaded on an SDS-8% polyacrylamide gel and
visualized by Coomassie brilliant blue staining. Lanes: 1, ACT; 2, ACTK58Q; 3, molecular weight markers.
Preparation of platelets. (i) Platelet preparation for cAMP assay. Platelets were prepared as described by Kitamura et al. (32, 33), with slight modification. Japanese White rabbits (3 to 4 kg) were bled (10 ml) into a 10-ml syringe containing 1/10 volume of 3.8% sodium citrate. The blood was centrifuged at 190 × g for 20 min, and the upper layer (platelet-rich plasma [PRP]) was isolated. Eventually, the lower layer, which still contained platelets, was resuspended in washing medium (135 mM NaCl, 4 mM glucose, 2 mM EDTA, 13 mM sodium acetate [pH 6.5]) and centrifuged at 190 × g for 20 min, and the upper layer was added to PRP. PRP or washing medium-containing PRP was then centrifuged again at 190 × g for 5 min to remove contaminating nonplatelet cells, and then the supernatant was centrifuged at 800 × g for 10 min to precipitate platelets. Precipitated platelets were resuspended in 10 ml of washing medium. The platelets were washed two times in washing medium by recentrifuging at 800 × g for 10 min and then once with suspension medium (135 mM NaCl, 5 mM glucose, 2 mM EDTA, 15 mM HEPES [pH 7.5]). The platelets were finally resuspended in 5 ml of suspension medium and kept at 37°C to prevent spontaneous aggregation. The final platelet preparation contained 1 nonplatelet cell per 2,000 to 10,000 platelets, which did not significantly affect cAMP measurements (data not shown).
(ii) Platelet preparation for aggregation assay. PRP was isolated as described above except that Na,K-Tris (137 mM NaCl, 5.4 mM KCl, 11 mM glucose, 25 mM Tris-HCl [pH 7.4]) was used instead of washing medium (31). PRP or Na,K-Tris-containing PRP was recentrifuged at 800 × g for 10 min, and precipitated platelets were resuspended in 450 µl of Na,K-Tris. Then 50 µl of 112 mM citrate (pH 6.0) was added to the platelet suspension, and the suspension was kept at 37°C to prevent spontaneous aggregation. The final preparation contained 1 nonplatelet cell (which did not contribute to aggregation) per at least 1,000 platelets.
Establishment and production of monoclonal antibody B7E11. The adenylate cyclase toxin fragment AC196-267, comprising aa 196 to 267 (corresponding to the calmodulin binding site of the catalytic domain), was expressed in E. coli and purified as described previously (39). Purified AC196-267 was kindly provided by Hélène Munier. Spleen cells of mice highly immunized with AC196-267 (a kind gift of Nicole Guiso) were fused with the murine fusion line SP2, and the specificity of antibody secreted by cloned hybridomas was identified by screening of culture supernatants in ACT and AC196-267 enzyme-linked immunosorbent assay (ELISA). For ELISA, the purified protein of interest (10 µg/ml; 100 µl/well) was directly coated overnight onto Maxisorp Microtiter plates (Nunc, Roskilde, Denmark) in alkaline carbonate buffer (pH 9.6). The wells were then blocked with phosphate-buffered saline (PBS) containing 2% bovine serum albumin for 4 h at room temperature. Hybridoma supernatants were incubated 2 h in the wells, followed by washing with PBS-bovine serum albumin containing 0.5% Tween 20. Bound antibody was detected by using goat anti-mouse antibody labeled with alkaline phosphatase and revealed with the substrate p-nitrophenyl phosphate (Sigma); plates were read at wavelength 405 nm. Antibody B7E11 was purified from hybridoma supernatant by protein A-agarose chromatography (HiTrap; Pharmacia, Uppsala, Sweden), purity was checked by SDS-PAGE, and the protein content of purified antibody was determined by the Pierce BCA protein assay.
cAMP assay. A 125-µl portion of platelet suspension was added to 125 µl of prewarmed (37°C) suspension medium containing 4 mM CaCl2, which gave a final concentration of 1 mM in the 250-µl incubation due to the chelating effect of EDTA in the platelet suspension. To avoid protein denaturation, appropriate concentrations of ACT and ACTK58Q were added to the medium immediately before addition of platelet suspension. Urea concentration in all incubations were equilibrated to 48 mM. The mixture was then incubated at 37°C, and cAMP was extracted by the method of Kitamura et al. (32, 33). Briefly, 1 ml of ethanol containing 0.15 M HCl was added to the incubation mixture, and platelets were immediately disrupted by sonication for 10 s with a model UP 50 H sonicator (Dr Hielscher GmbH, Teltow, Germany). The sonicated extract was heated at 100°C for 10 min, and then solvent was evaporated. Residues were redissolved in 1 ml of 50 mM sodium acetate buffer (pH 6.2), and 10 to 100 µl was subjected to radioimmunoassay (1) using rabbit anti-cAMP polyclonal antiserum (ICN Biochemicals, Costa Mesa, Calif.). Sixty percent of cAMP had already accumulated during first 5 min of a 15-min incubation period and thus was already present at a level about 170 times higher than physiological cAMP levels (data not shown). Thus, to avoid spontaneous platelet aggregation during unnecessarily prolonged incubation periods, 5 min of ACT treatment was used for cAMP and aggregation assays throughout this study.
Platelet aggregometry. ADP- or thrombin-induced platelet aggregation was expressed as decrease of absorbance (4) (increase of light transmittance) of platelet suspension at 700 nm. Fifty microliters of platelet suspension was added to an aggregometer cuvette (no. 3121; SSR Engineering Co., Tokyo, Japan) containing 450 µl of Na,K-Tris supplemented with 1 mM CaCl2. Rabbit fibrinogen (Sigma Chemical, St. Louis, Mo.) was added to 1 mg/ml for ADP-induced aggregation assays. ACT, ACTK58Q, dibutyryl-cAMP, or forskolin was then added, and the mixture was incubated at 37°C for 5 min. The urea concentration in the incubation mixture was kept below 45 mM, as described in the figure legends. After incubation, the cuvettes were placed in the incubation chambers of the two-chamber aggregometer (model PAT-2M; SSR Engineering), and absorbance of this initial mixture was set as 100%; absorbance of Na,K-Tris was set as 0%. Fifty microliters of 100 µM ADP or 2 U/ml of thrombin (Sigma) per incubation (final concentrations of 10 µM or 0.2 U/ml, respectively) was added gently but rapidly, using a 50-µl microsyringe, and the change in absorbance was monitored and recorded with a two-pen recorder (model U-228; Nippon Denshi Kagaku, Kyoto, Japan).
Extensive care was taken for antibody-blocking experiment in order to establish experimental conditions which allow a proper antigen-antibody reaction without impairing the aggregation potential of platelets. Because the internalization of ACT into target cells is considered to be very rapid, antibody molecules are not likely to be able to complete their binding to ACT molecules before ACT internalizes across the platelet cell membrane. On the other hand, a frequently used method, such as preincubation of antibody with ACT prior to addition of the platelet suspension, could not be successfully employed either, because of nonspecific inactivation of ACT due to self-aggregation in the absence of denaturing agents such as 8 M urea. Thus, during antibody treatment, translocation of membrane-inserted ACT molecules into platelets was prevented by not adding calcium ions (43) (but not by chelating) into the Na,K-Tris incubation medium. This mixture was incubated at 37°C for 8 min, and then a 1/20 volume of 20 mM CaCl2 was added to allow ACT internalization. After 5 min of incubation at 37°C, ADP was added and changes in absorbance at 700 nm were recorded.Bleeding time assay. The bleeding time assay was performed as described by Dejana et al. (10) and others (40, 50). Briefly, ACT or ACTK58Q (100 µg/mouse) was injected intravenously to female Slc: ddY mice (47) (8 weeks old) at the base of the tail. Thirty minutes after injection, the tail tip (2 mm) was cut under pentobarbital (Nembutal; 50 mg/kg of body weight, Abbott Laboratories, Chicago, Ill.) anesthesia, and the tail was immediately immersed in PBS (37°C). Bleeding time was measured from the time of tail transsection to an abrupt stopping of bleeding without rebleeding within 30 s. Bleeding times exceeding 15 min were recorded as 900 s.
| |
RESULTS |
|---|
|
|
|---|
Platelet cAMP accumulation by ACT. To examine whether ACT is able to act on rabbit platelets, we first measured the effect of ACT on platelet intracellular cAMP levels. Rabbit platelets (1.2 × 108/ml) were treated with 0.3 to 10 µg/ml (equivalent to a catalytic activity of 0.12 to 4.0 µmol of cAMP/min) of DEAE-purified ACT in 250 µl of suspension medium containing 1 mM CaCl2 at 37°C for 5 min, and then intracellular cAMP was extracted by ultrasonic cell disruption in ethanol-HCl and measured by radioimmunoassay (Fig. 2). ACT induced an increase in cAMP level in platelets dose dependently, up to 5 nmol/109 platelets at 10 µg/ml. In contrast, no significant increase of intracellular cAMP level was induced by 10 µg of ACT per ml in the absence of calcium, which is known to be required for internalization of the toxin into cells (43). The catalytically inactive mutant ACTK58Q did not show a significant increase of intracellular cAMP even at a concentration as high as 10 µg/ml, indicating that the cAMP increase by catalytically active ACT is a consequence of its internalization into platelets. Forskolin (10 µM) induced a slight but significant increase of intracellular cAMP compared to platelets containing urea, ACTK58Q, or ACT (without CaCl2) (Fig. 2). As cAMP levels in urea-treated platelets were significantly higher than those of untreated platelets (Fig. 2), urea treatment provided more suitable controls.
|
Suppression of ADP-induced platelet aggregation by ACT. We then investigated the effects of ACT on the aggregation potency of rabbit platelets through an increase of intracellular cAMP levels.
Rabbit platelets (1.5 × 108/ml) were resuspended in Na,K-Tris supplemented with 1 mM CaCl2, aggregation was initiated by the addition of 10 µM ADP, and absorbance of the platelet suspension was monitored with a platelet aggregometer. A dramatic decrease in absorbance due to aggregation was observed within 1 min after addition of ADP to control (buffered 8 M urea-treated) platelet suspension, which reflects the normal reactivity of the platelets to ADP (Fig. 3). Treatment of platelets with ACT (3 µg/ml; equivalent to a catalytic activity of 1.2 µmol of cAMP/min) for 5 min at 37°C, prior to addition of 10 µM ADP, completely suppressed the aggregation (Fig. 3). This suppression was also observed by forskolin (10 µM) and dibutyryl-cAMP (1 mM) treatments (Fig. 3). In contrast, the catalytically inactive mutant ACTK58Q at 3 µg/ml (Fig. 3) or 10 µg/ml (data not shown) did not significantly suppress ADP-induced aggregation, suggesting that the impairment of platelet function resulted from accumulation of intracellular cAMP. There was no significant difference between buffered 8 M urea-treated and untreated controls (data not shown), which was compatible with the results of Camici et al. (6).
|
|
|
Suppression of thrombin-induced platelet aggregation by ACT. Figure 6 shows the effect of ACT on thrombin-induced platelet aggregation. Thrombin (0.2 U/ml) more strongly induced aggregation on control platelets, and this aggregation was suppressed by 3 µg of ACT per ml. Stronger suppression was achieved by treatment with higher concentrations of ACT (up to 10 µg/ml), again showing a clear dose dependence, which may reflect higher intracellular cAMP accumulation evoked by 10 µg/ml than by 3 µg/ml, as shown in Fig. 2. The same concentration (10 µg/ml) of a catalytically inactive mutant ACTK58Q did not suppress thrombin-induced aggregation. There was no significant difference between buffered 8 M urea-treated and untreated controls (data not shown). These results clearly indicate that the suppression of ADP- and thrombin-induced platelet aggregation was due to the catalytic activity of ACT through increase of intracellular cAMP.
|
In vivo effect of ACT on mouse bleeding time.
We then examined
whether this suppressive effect of ACT on platelet aggregation would
occur in vivo in mice. ACT and ACTK58Q were injected intravenously into
the tail vein of mice at the base of the tail, to avoid destruction of
tail tissue near the tail tip. Thirty minutes after injection, the tail
tip (2 mm) was cut with a sharp razor blade under pentobarbital
anesthesia, the tail was immediately immersed in PBS (37°C), and
bleeding times were measured for eight to nine mice per group.
Significance of difference between groups was confirmed with Student's
t test. Table 1 shows the
bleeding times of ACT-treated, ACTK58Q-treated, and control (buffered 8 M urea-treated) mice. The result clearly show a significant
prolongation of bleeding time by ACT treatment, compared to
ACTK58Q-treated and control groups, indicating the in vivo suppressive
activity of ACT on blood coagulation.
|
| |
DISCUSSION |
|---|
|
|
|---|
We observed that ACT suppresses ADP- and thrombin-induced aggregation of rabbit platelets in vitro. We also observed a large delay of bleeding time in vivo in ACT-treated mice, probably resulting from the suppression of platelet aggregation. The in vitro suppression was dependent on ACT concentration in the incubation medium and reached complete suppression of ADP-induced aggregation at a high concentration such as 3 µg/ml (Fig. 3). The suppression was accompanied by a marked increase in intracellular cAMP levels (Fig. 2), and the catalytically inactive mutant ACTK58Q showed no such suppressive effect (Fig. 3). As cAMP is a widely recognized suppressor of platelet aggregation (25, 37, 46, 56), the results clearly indicate that ACT suppresses platelet aggregation by increasing intracellular cAMP through its catalytic activity. This observation is also supported by the fact that the suppressive effect was diminished by adding the anti-ACT monoclonal antibody B7E11, directed against the calmodulin binding site in the catalytic domain of ACT (Fig. 4). This antibody is also able to inhibit the internalization of ACT into sheep erythrocytes (data not shown), suggesting that the partial inhibition of ACT action by this antibody was due to blocking of ACT entry into platelets but not to inhibition of the catalytic activity of ACT. Preliminary experiments indicate that this antibody does not exert a significant inhibitory effect on the adenylate cyclase catalytic activity of ACT. This is often the case; among a large number of monoclonal or polyclonal antibodies (raised in rabbits, guinea pigs, or mice) against ACT, inhibition of adenylate cyclase activity was observed only once (51a).
cAMP is known to suppress both ADP- and thrombin-induced platelet aggregation (25, 37, 46, 56). ADP and thrombin trigger platelet activation by release of calcium from intracellular pools, through activation of phospholipase C and subsequent release of inositol 1,4,5-triphosphate (15). Alternatively, ADP is considered to stimulate calcium influx by opening of calcium channels which are coupled with membrane ADP receptors (15). Various mechanisms have been proposed for cAMP-mediated platelet inactivation, including (i) promotion of calcium efflux through activation of plasma membrane Ca2+-ATPase by cAMP-dependent protein kinase (9, 29), (ii) reduction of phospholipase C/inositol 1,4,5-triphosphate-mediated calcium mobilization from intracellular stores by interacting with phospholipase C (15), and (iii) reduction of the binding and response of platelets to thrombin (36). Although the mechanism of cAMP-mediated suppression of platelet aggregation has not yet been clearly elucidated, our results are fully consistent with all of these proposed mechanisms, as ACT simply produces supraphysiological amount of intracellular cAMP in platelets. In contrast, the increase of intracellular cAMP by 10 µM forskolin was significant but lower than that induced by 0.3 µg of ACT per ml (Fig. 2), although the same concentration of forskolin completely suppressed ADP-induced platelet aggregation (Fig. 3) whereas 0.2 or 0.5 µg ACT per ml did not (Fig. 5). The reason for this discrepancy remains unclear. One possible explanation could be the difference between the mechanisms of action of forskolin and ACT. Forskolin increases cAMP concentration by activating an exquisitely regulated cellular adenylate cyclase (29), while the calmodulin-activated invasive ACT catalyzes high-level synthesis of cAMP, which might alter cellular physiology in a different way.
Both ADP- and thrombin-induced platelet aggregation were suppressed by ACT in a dose-dependent manner. However, the ADP-induced aggregation appeared to be more sensitive to the suppressive effects of ACT than that induced by thrombin under our experimental conditions. An ACT concentration of 3 µg/ml was sufficient to completely suppress the ADP-induced platelet aggregation (Fig. 3), while concentrations as high as 10 µg/ml did not bring about a complete suppression of thrombin-induced aggregation (Fig. 6). This could be due to the difference between the mechanisms of platelet aggregation induced by these two agents. In fact, ADP-induced aggregation was partially reversible (data not shown), and the maximum reduction of absorbance did not exceed 50% (Fig. 3). In contrast, thrombin-induced aggregation was biphasic and irreversible, forming a cluster tightly attached to the stirring bar in the aggregometer cuvette, and the maximum reduction of absorbance reached almost 100% (Fig. 6). In addition, these agents may not be equally effective as inducers of platelet aggregation at the concentrations used in this study.
Numerous experimental data (7, 23, 30, 41) have shown that ACT serves to disable the host defense function of immune effector cells during the early stage of B. pertussis infection. In addition to these actions, we reported here the effects of ACT on platelet aggregation and bleeding time not only in vitro but also in vivo; however, the significance of these biological activities in pertussis disease is not yet clear. Pertussis disease is often accompanied by complications such as subconjunctival hemorrhage, skin petechiae, epistasis, hemoptysis, and occasionally intracranial hemorrhage (52). These symptoms are generally regarded as a result of venous congestion (52); however, these hemorrhagic symptoms resemble platelet dysfunction and could also be attributed, at least in part, to ACT. On the other hand, ACT is not considered to circulate in the bloodstream in clinical pertussis cases, as evidenced by the facts that (i) viable B. pertussis bacteria have been very rarely detected in circulating blood of patients (28, 52) and (ii) the presence of ACT in circulating blood has, to our knowledge, never been reported. To clarify whether ACT contributes to the symptoms of pertussis, further experimental evidence is needed. In vivo and in vitro analysis of platelet functions of patients and experimentally infected animals would help to elucidate the role of ACT in pertussis disease.
| |
ACKNOWLEDGMENTS |
|---|
We thank Agnes Ullmann, Hiroko Sato, and Yoshichika Arakawa for critical reading of the manuscript and continuous encouragement throughout this work.
N.H. was supported by the European Community (Human Capital and Mobility Programme).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Bacterial and Blood Products, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan. Phone: 81-42-561-0771, ext. 363. Fax: 81-42-561-7173. E-mail: miwaki{at}nih.go.jp.
Present address: INSERM U 332, Institut Cochin de
Génétique Moléculaire, 75014 Paris, France.
Editor: J. T. Barbieri
| |
REFERENCES |
|---|
|
|
|---|
| 1. |
Bellalou, J.,
H. Sakamoto,
D. Ladant,
C. Geoffroy, and A. Ullmann.
1990.
Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase.
Infect. Immun.
58:3242-3247 |
| 2. |
Benz, R.,
E. Maier,
D. Ladant,
A. Ullmann, and P. ebo.
1994.
Adenylate cyclase toxin of Bordetella pertussis: evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli.
J. Biol. Chem.
269:27231-27239 |
| 3. |
Betsou, F.,
P. ebo, and N. Guiso.
1993.
CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin.
Infect. Immun.
61:3583-3589 |
| 4. | Born, G. V. R. 1962. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194:927-929[Medline]. |
| 5. | Brownlie, R. M., J. G. Coote, R. Parton, J. E. Schultz, A. Rogel, and E. Hanski. 1988. Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis. Microb. Pathog. 4:335-344[Medline]. |
| 6. | Camici, M., L. Evangelisti, and M. Raspolli-Galletti. 1986. The effect of oxalic acid on the aggregability of human platelet rich plasma. Prostaglandins Leukot. Med. 21:107-110[Medline]. |
| 7. |
Confer, D. L., and J. W. Eaton.
1982.
Phagocyte impotence caused by an invasive bacterial adenylate cyclase.
Science
217:948-950 |
| 8. | Coote, J. G. 1992. Structural and functional relationships among the RTX toxin determinants of Gram-negative bacteria. FEMS Microbiol. Rev. 88:137-162. |
| 9. |
Dean, W. L.,
D. Chen,
P. C. Brandt, and T. C. Vanaman.
1997.
Regulation of platelet plasma membrane Ca2+-ATPase by cAMP-dependent and tyrosine phosphorylation.
J. Biol. Chem.
272:15113-15119 |
| 10. | Dejana, E., A. Calloni, A. Quintana, and G. de Gaetano. 1979. Bleeding time in laboratory animals. II. A comparison of different assay conditions in rats. Thromb. Res. 15:191-197[Medline]. |
| 11. | Donovan, M. G., and D. R. Storm. 1990. Evidence that the adenylate cyclase secreted from Bordetella pertussis does not enter animal cells by receptor-mediated endocytosis. J. Cell Physiol. 145:444-449[Medline]. |
| 12. | Ehrmann, I. E., M. C. Gray, V. M. Gordon, L. S. Gray, and E. L. Hewlett. 1991. Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis. FEBS Lett. 278:79-83[Medline]. |
| 13. | Ehrmann, I. E., A. A. Weiss, M. S. Goodwin, M. C. Gray, E. Barry, and E. L. Hewlett. 1992. Enzymatic activity of adenylate cyclase toxin from Bordetella pertussis is not required for hemolysis. FEBS Lett. 304:51-56[Medline]. |
| 14. | Friedman, E., Z. Farfel, and E. Hanski. 1987. The invasive adenylate cyclase of Bordetella pertussis: properties and penetration kinetics. Biochem. J. 243:145-151[Medline]. |
| 15. | Geiger, J., and U. Walter. 1993. Properties and regulation of human platelet cation channels. EXS 66:281-288[Medline]. |
| 16. | Gentile, F., A. Raptis, L. G. Knipling, and J. Wolff. 1988. Bordetella pertussis adenylate cyclase: penetration into host cells. Eur. J. Biochem. 175:447-453[Medline]. |
| 17. | Glaser, P., A. Elmaoglou-Lazaridou, E. Krin, D. Ladant, O. Bârzu, and A. Danchin. 1989. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis. EMBO J. 8:967-972[Medline]. |
| 18. | Glaser, P., D. Ladant, O. Sezer, F. Pichot, A. Ullmann, and A. Danchin. 1988. The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol. Microbiol. 2:19-30[Medline]. |
| 19. |
Goodwin, M. S., and A. A. Weiss.
1990.
Adenylate cyclase toxin is critical for colonization and Pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice.
Infect. Immun.
58:3445-3447 |
| 20. |
Gordon, V. M.,
S. H. Leppla, and E. L. Hewlett.
1988.
Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin.
Infect. Immun.
56:1066-1069 |
| 21. |
Gordon, V. M.,
W. W. Young, Jr.,
S. M. Lechler,
M. C. Gray,
S. H. Leppla, and E. L. Hewlett.
1989.
Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells.
J. Biol. Chem.
264:14792-14796 |
| 22. |
Gross, M. K.,
D. C. Au,
A. L. Smith, and D. R. Storm.
1992.
Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.
Proc. Natl. Acad. Sci. USA
89:4898-4902 |
| 23. |
Gueirard, P.,
A. Druilhe,
M. Pretolani, and N. Guiso.
1998.
Role of adenylate cyclase-hemolysin in alveolar macrophage apoptosis during Bordetella pertussis infection in vivo.
Infect. Immun.
66:1718-1725 |
| 24. | Hanski, E., and Z. Farfel. 1985. Bordetella pertussis invasive adenylate cyclase: partial resolution and properties of its cellular penetration. J. Biol. Chem. 290:5526-5532. |
| 25. | Haslam, R. J., M. M. L. Davidson, T. Davies, J. A. Lynham, and M. D. McClenaghan. 1978. Regulation of blood platelet function by cyclic nucleotides. Adv. Cyclic Nucleotide Res. 9:533-552[Medline]. |
| 26. |
Hewlett, E. L.,
L. Gray,
M. Allietta,
I. E. Ehrmann,
V. M. Gordon, and M. C. Gray.
1991.
Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity.
J. Biol. Chem.
266:17503-17508 |
| 27. |
Iwaki, M.,
A. Ullmann, and P. ebo.
1995.
Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin.
Mol. Microbiol.
17:1015-1024[Medline].
|
| 28. |
Janda, W. M.,
E. Santos,
J. Stevens,
D. Celig,
L. Terrile, and P. C. Schreckenberger.
1994.
Unexpected isolation of Bordetella pertussis from a blood culture.
J. Clin. Microbiol.
32:2851-2853 |
| 29. | Johansson, J. S., L. E. Nied, and D. H. Haynes. 1992. Cyclic AMP stimulates Ca2+-ATPase-mediated Ca2+ extrusion from human platelets. Biochim. Biophys. Acta 1105:19-28[Medline]. |
| 30. |
Khelef, N.,
A. Zychlinsky, and N. Guiso.
1993.
Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin.
Infect. Immun.
61:4064-4070 |
| 31. | Kitagawa, S., K. Kotani, and F. Kametani. 1990. Inhibitory mechanism of cis-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca2+ mobilization, cyclic AMP levels and membrane fluidity. Biochim. Biophys. Acta 1054:114-118[Medline]. |
| 32. | Kitamura, K., K. Kangawa, M. Kawamoto, Y. Ichiki, H. Matsuo, and T. Eto. 1992. Isolation and characterization of peptides which act on rat platelets, from a pheochromocytoma. Biochem. Biophys. Res. Commun. 185:134-141[Medline]. |
| 33. | Kitamura, K., K. Kangawa, M. Kawamoto, Y. Ichiki, S. Nakamura, H. Matsuo, and T. Eto. 1993. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192:553-560[Medline]. |
| 34. |
Ladant, D.,
S. Michelson,
R. S. Sarfati,
A.-M. Gilles,
R. Predeleanu, and O. Bârzu.
1989.
Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase.
J. Biol. Chem.
264:4015-4020 |
| 35. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]. |
| 36. |
Lerea, K. M.,
J. A. Glomset, and E. G. Krebs.
1987.
Agents that elevate cAMP levels in platelets decrease thrombin binding.
J. Biol. Chem.
262:282-288 |
| 37. | Marquis, N. R., R. L. Vigdahl, and P. A. Tavormina. 1969. Platelet aggregation. I. Regulation by cyclic AMP and prostaglandin E1. Biochem. Biophys. Res. Commun. 36:965-972[Medline]. |
| 38. |
Mouallem, M.,
Z. Farfel, and E. Hanski.
1990.
Bordetella pertussis adenylate cyclase toxin: intoxication of host cells by bacterial invasion.
Infect. Immun.
58:3759-3764 |
| 39. | Munier, H., A. Bouhss, A.-M. Gilles, N. Palibroda, O. Bârzu, J. Mispelter, and C. T. Craescu. 1995. Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase. Arch. Biochem. Biophys. 320:224-235[Medline]. |
| 40. | Novak, E. K., H. O. Sweet, M. Prochazka, M. Parentis, R. Soble, M. Reddington, A. Cairo, and R. T. Swank. 1988. Cocoa: a new mouse model for platelet storage pool deficiency. Br. J. Haematol. 69:371-378[Medline]. |
| 41. | Pearson, R. D., P. Symes, M. Conboy, A. A. Weiss, and E. L. Hewlett. 1987. Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin. J. Immunol. 139:2749-2754[Abstract]. |
| 42. |
Raptis, A.,
L. Knipling, and J. Wolff.
1989.
Dissociation of catalytic and invasive activities of Bordetella pertussis adenylate cyclase.
Infect. Immun.
57:1725-1730 |
| 43. |
Rogel, A., and E. Hanski.
1992.
Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes. Translocation of the toxin across the membrane.
J. Biol. Chem.
267:22599-22605 |
| 44. |
Rogel, A.,
R. Meller, and E. Hanski.
1991.
Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.
J. Biol. Chem.
266:3154-3161 |
| 45. |
Sakamoto, H.,
J. Bellalou,
P. ebo, and D. Ladant.
1992.
Bordetella pertussis adenylate cyclase toxin: structural and functional independence of the catalytic and hemolytic activities.
J. Biol. Chem.
267:13598-13602 |
| 46. | Salzman, E. W., E. B. Rubino, and R. V. Sims. 1970. Cyclic 3',5'-adenosine monophosphate in human blood platelets. III. The role of cyclic AMP in platelet aggregation. Ser. Haemat. 3:100-113. |
| 47. |
Sato, H., and Y. Sato.
1984.
Bordetella pertussis infection in mice: correlation of specific antibodies against two antigens, pertussis toxin, and filamentous hemagglutinin with mouse protectivity in a intracerebral or aerosol challenge system.
Infect. Immun.
46:415-421 |
| 48. | Sebo, P., P. Glaser, H. Sakamoto, and A. Ullmann. 1991. High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system. Gene 104:19-24[Medline]. |
| 49. | Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85[Medline]. |
| 50. | Swank, R. T., M. Reddington, and E. K. Novak. 1996. Inherited prolonged bleeding time and platelet storage pool deficiency in the Subtle Gray (sut) mouse. Lab. Anim. Sci. 46:56-60[Medline]. |
| 51. |
Szabo, G.,
M. C. Gray, and E. L. Hewlett.
1994.
Adenylate cyclase toxin from Bordetella pertussis produces ion conductance across artificial lipid bilayers in a calcium- and polarity-dependent manner.
J. Biol. Chem.
269:22496-22499 |
| 51a. | Ullmann, A. Personal communication. |
| 52. | Walker, E. 1988. Clinical aspects of pertussis, p. 273-282. In A. C. Wardraw, and R. Parton (ed.), Pathogenesis and immunity of pertussis. John Wiley & Sons, Chichester, England. |
| 53. | Weiss, A. A., E. L. Hewlett, G. A. Myers, and S. Falkow. 1984. Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis. J. Infect. Dis. 150:219-222[Medline]. |
| 54. |
Westrop, G. D.,
G. Campbell,
Y. Kazi,
B. Billcliffe,
J. G. Coote,
R. Parton,
J. H. Freer, and J. G. Edwards.
1994.
A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectin-stimulated spreading of BHK21 cells.
Microbiology
140:245-253 |
| 55. |
Wolff, J.,
G. H. Cook,
A. R. Goldhammer, and S. A. Berkowitz.
1980.
Calmodulin activates prokaryotic adenylate cyclase.
Proc. Natl. Acad. Sci. USA
77:3841-3844 |
| 56. | Zieve, P. D., and W. B. Greenough, III. 1969. Adenyl cyclase in human platelets: activity and responsiveness. Biochem. Biophys. Res. Commun. 35:462-466[Medline]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»