INFECTION AND IMMUNITY, Nov. 1990, p. 3621-3626 0019-9567/90/113621-06$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 58, No. 11

Adenylate Cyclase Toxin of Bordetella pertussis: Production, Purification, and Partial Characterization MARK S. LEUSCH, SUZANNE PAULAITIS,

RICHARD L. FRIEDMAN* Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona 85724 AND

Received 12 April 1990/Accepted 21 August 1990

Bordetella pertussis produces a number of virulence determinants which contribute to its pathogenicity. One factor, the adenylate cyclase toxin (ACT), has been suggested to directly penetrate human phagocytes and disrupt their normal function by direct production of intracellular cyclic AMP (cAMP). Experiments evaluating the production of cell-associated ACT in liquid cultures of B. pertussis 504 demonstrated that the greatest activity was observed during mid-log-phase growth. Urea extracts of cells harvested during the time of maximal ACT production have been used to purify the toxin with both biological and enzymatic activities. ACT is a protein with an apparent molecular mass of 220 kDa and an isoelectric point of 7.0. The specific activity of purified ACT is 17,000 ,umol of cAMP formed per mg per min. The biological specific activity of purified ACT is 6,250 nmol of intracellular cAMP formed per mg per min in 2 x 106 S49 lymphoma cells per ml. Preparations containing 8 ,ug of ACT completely abrogated the chemiluminescence

human neutrophils



of 2 x 106


Pertussis is the disease in humans caused by the gramnegative coccobacillus Bordetella pertussis (18). The organism is known to produce a number of virulence determinants, among which is the adenylate cyclase toxin (ACT). The biological activity of ACT has been demonstrated with urea extracts of virulent whole-cell B. pertussis. These extracts inhibited the production of superoxide in human neutrophils and macrophages (2). The biological activity of ACT is mediated directly through its enzymatic function of converting host ATP to cyclic AMP (cAMP). A unique aspect of B. pertussis ACT is that the enzymatic activity can be enhanced by the eucaryotic calcium-dependent regulatory protein calmodulin (27). This enhancement of activity results in unregulated production of intracellular cAMP. The majority of ACT has been shown to be cell associated, since trypsin treatment of virulent whole-cell B. pertussis results in a rapid loss of ACT enzymatic activity (11). This suggested that ACT was in the outer membrane of the cell. Studies evaluating production of ACT enzymatic activity in liquid culture suggested that the majority of the cell-associated ACT appeared during early logarithmic growth (3). In order to study the nature of ACT and its role in pathogenesis, it is important to purify toxin with intact biological and enzymatic activities. The purification of B. pertussis ACT has been attempted by numerous investigators since the toxin was first identified. The typical starting material for purification, whether from urea-extracted whole cells or from culture supernatant, has been obtained from cultures grown for a minimum of 20 to 24 h (10, 12, 13, 15, 19, 20, 27). Starting material from cultures as old as 48 h has also been used (22). These studies gave extremely low protein recoveries and report a range of molecular masses from 43 to 700 kDa. Evaluation of these preparations often focused only on the enzymatic activity (12, 13, 15, 27). Previously, Friedman described purification of a 60-kDa protein which had both ACT biological and enzymatic activities (4). These preparations contained a minor protein

contaminant of about 200 kDa. Through the method described in this paper, we have separated the 220- and 60-kDa proteins and demonstrated that both desired ACT properties were associated exclusively with the 220-kDa protein. The molecular mass of this protein is in close agreement with the size calculated on the basis of the gene sequence (7). In the present studies, growth curve experiments were performed to determine the time of maximal cell-associated ACT production. Using this information, we were able to purify a substantial quantity of native ACT which contained both the biological and enzymatic properties. A recent report described that ACT biological activity was associated with a single 200-kDa protein (23). We have substantiated this claim as well as demonstrated that ACT is a 220-kDa protein with both biological and enzymatic activity. MATERIALS AND METHODS

Bacteria and growth curve. B. pertussis 504, a virulent wild-type isolate, and BP353, a TnS filamentous hemagglutinin (FHA)-negative mutant (a generous gift from David Relman, Stanford University) were stored in Stainer-Scholte medium containing 50% glycerol at -70°C. Bacteria were plated on charcoal agar (Difco Laboratories, Detroit, Mich.) supplemented with 10% sheep blood and grown at 37°C for 36 to 40 h. Growth from a confluent plate was used to seed starter cultures in 100 ml of Stainer-Scholte medium which were grown on a rotary shaker at 37°C for 24 h. At specified time points, 1-ml aliquots of cells were removed and cell density was measured at 650 nm. Aliquots were centrifuged, and the cell pellets were washed with 50 mM Tris (pH 7.6S50 mM NaCl (TN), suspended in 1 ml of TN, and assayed for ACT enzymatic activity. In experiments evaluating biological activity, washed cell pellets were extracted with TN containing 6 M urea and dialyzed against TN, and 20 ,ug (in 50 ,ul) was assayed for inhibition of neutrophil chemiluminescence (CL) and for increased cAMP levels in S49 lymphoma cells. Assays to determine the enzymatic activity of these urea extracts were also done for comparison.


Cultures of B. pertussis for purification of ACT were obtained by inoculating flasks containing 400 ml of Stainer-

Corresponding author. 3621



Scholte medium with 50 ml of BP504 from 12- to 14-h starter cultures. Cells were grown at 37°C for 10 h, harvested by centrifugation at 10,000 x g at 4°C for 10 min, and washed with TN. The resultant pellet was weighed and homogenized with an Ultra-turrax (Tekmar Co., Cincinnati, Ohio) with a setting not exceeding 30 in 50 mM Tris (pH 7.6)-50 mM EDTA at a cell concentration of 0.1 g of cells per ml. Lysozyme (Sigma, St. Louis, Mo.) and phenylmethylsulfonyl fluoride (Sigma) were added to final concentrations of 0.25 mg/ml and 0.25 mM, respectively, and the mixture was incubated on ice for 20 min. Cells were then centrifuged as before, and the pellet was homogenized in 50 mM Tris (pH 7.6)-150 mM NaCl-1 mM EDTA-0.25 mM phenylmethylsulfonyl fluoride to a density of 0.1 g of cells per ml. Solid urea (Ultra pure; Fluka, Ronkonkoma, N.Y.) was added to a final concentration of 6 M, and the resultant extract was stored for a minimum of 18 h at -70°C. Chromatography. Unless otherwise indicated, all manipulations were performed at 4°C. Prior to chromatography, urea extracts of BP504 were centrifuged at 45,000 x g for 20 min to remove cell debris. The urea extract (60 ml) was dialyzed against several changes of 10 mM sodium phosphate buffer (pH 8.0). The extract was diluted fivefold and passed over a 185-ml spheroidal hydroxylapatite (HA) (Gallard-Schlesinger, Carle Place, N.Y.) column at 28 ml/h. The column was washed with 10 mM phosphate buffer (pH 8.0) until a baseline absorbance (A280) was obtained. Protein bound to the column was eluted with 100 mM phosphate buffer (pH 8.0) and detected by measuring the A280. Individual peak fractions were assayed for ACT enzymatic activity and pooled. The HA active pool (40 ml) was dialyzed against several changes of 50 mM Tris (pH 7.6)-S150 mM NaCl-5 mM MgCl2-0.1 mM CaCl2 (buffer A). The dialysate was diluted in buffer A, and solid 3-[(3-cholamidopropyl)-dimethylammonia]-1-propanesulfonate (CHAPS) was added to a final concentration of 2 mM. The protein was passed over a 10-ml calmodulin-Sepharose (Pharmacia, Piscataway, N.J.) column equilibrated with buffer A containing 2 mM CHAPS at 12 mi/h. The column was washed with 3 bed volumes of the same buffer followed by 3 bed volumes of buffer A. The column was then eluted with buffer A containing 6 M urea. Protein elution was detected by measuring the A280, and peak fractions were assayed for both ACT biological and enzymatic activities. Protein concentrations were determined by BCA protein assay (Pierce, Rockford, Ill.). In certain instances, bovine serum albumin (BSA; Sigma) was added to the purified preparations at a final concentration of 0.1 mg/ml. Adenylate cyclase enzyme assay. All enzyme assays were performed by a modified version of the method outlined by White (26). A 40-,I sample to be tested was preincubated with 3 RI of calmodulin (0.5 mg/ml; Pharmacia) at 30°C for 10 min. The reaction was initiated by the addition of 40 ,ul of ATP mixture (40 mM Tris [pH 7.6], 10 mM MgCi2, 0.4 mM CaCIl, 2 mM ATP, 0.2% BSA) containing 0.5 p.Ci of [a-32P]ATP (3,000 Ci/mmol; NEN, Boston, Mass.) per reaction. Reactions were conducted at 30°C for 25 min and then stopped by the addition of 200 ,u1 of 0.5 N HCI, and the pH was restored by the addition of 300 ,ul of 1.5 M imidazole. cAMP was recovered from 1-g alumina columns equilibrated with 100 mM imidazole (pH 7.6). Enzyme activity was reported in units, where 1 U equals 1 ,umol of cAMP formed per min. CL inhibition assay. Isolation of polymorphonuclear leukocytes and the CL inhibition assay were performed accord-


ing to the method of Friedman et al. (5). Viability of neutrophils was determined following incubation with ACT preparations by trypan blue exclusion. Activity was reported as the percent inhibition relative to the CL level of control (untreated) neutrophils. cAMP determination with S49 lymphoma cells. S49 lymphoma cells were a generous gift from Erik Hewlett, Departments of Medicine and Pharmacology, University of Virginia, Charlottesville. Cells were grown in suspension culture in Dulbecco modified Eagle medium (Sigma) containing 10% horse serum at 37°C in a 5% CO2 environment. Cells were harvested and resuspended in Dulbecco modified Eagle medium containing 0.2 mM isobutyl-methyl xanthine (Sigma) at a density of 2 x 106 cells per ml. Assays were initiated when 450 RI of cells was incubated with 50 p.1 of sample at 37°C for 1 h with rotation. Positive-control cells were incubated with 50 p.1 of 10 p.M forskolin. Negative-control cells were incubated with buffer alone. Cells were harvested by centrifugation for 10 min at 300 x g, and the supernatant was carefully aspirated. The pellets were suspended in 150 p.1 of 50 mM Tris (pH 7.5)-4 mM EDTA (TE buffer) and boiled for 5 min, and the cell lysate was centrifuged as described above for 2 min. The supernatants (50 pl.) were assayed in duplicate for the presence of cAMP by a modified version of the method described by Gilman (6). Samples were incubated with 50 p.1 of 20-pmol/ml 2,8,-3H-cAMP (32.8 Ci/mmol; NEN) and 100 p.1 of cAMP-dependent protein kinase (bovine heart; Sigma) on ice at 4°C for 2 h. Protein kinase was prepared as a stock at 60 p.g/ml in TE buffer containing 2% BSA (radioimmunoassay grade; Sigma). Reactions were terminated by the addition of 100 p.1 of activated carbon (Ultra SX; Norit, Amersfoort, The Netherlands) in TE buffer containing 2% BSA. Samples were centrifuged for 45 s, and 200 p.l of supernatant was removed and counted. Raw counts per minute were corrected by subtraction of blank counts per minute. A standard concentration curve ranging from 2.5 to 500 pmol/ml was established by plotting B/Bo (percent) versus picomoles of cAMP, where B is equal to the corrected value of the sample and Bo is the corrected value for the 0-pmol/ml standard. Sample B/Bo values were extrapolated to the curve to quantitate the concentration of cAMP. Biological activity was reported in units, where 1 U equals 1 nmol of cAMP formed per min. Electrophoresis. All sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the method of Blackshear (1), with 7.5 to 15% acrylamide gradient gels. Prestained molecular weight standards (BRL, Gaithersburg, Md.) were used to estimate protein molecular weights. Gels were silver stained by the method of Morrissey (16). Liquid-phase isoelectric focusing was performed with a Rotofor cell (Bio-Rad, Richmond, Calif.). Several calmodulin-Sepharose active pools were combined with 2% (wt/vol) 40% ampholines (pH 3.5 to 10; LKB, Bromma, Sweden). Proteins were separated for 5 h at full voltage and current (limited by constant power of 12 W) and then recovered by vacuum aspiration. After the pH gradient was recorded, the samples were dialyzed and assayed for ACT enzymatic activity. RESULTS Production of cell-associated ACT in liquid culture of BP504. A time course experiment to evaluate cell-associated ACT was performed in order to obtain whole-cell extracts from cultures during their peak time of ACT production. The


VOL. 58, 1990 2.0



0 U) 0







"I 0





0 CL



M 0.5

300 CD)




12 16 Time (hrs)




B a


-1 750

E D 0

0 E

600 H




400 I-

.0 E




-1 250

200 IOL



' 4


' 8 6 Time (hrs)

J 10



FIG. 1. (A) Production of cell-associated ACT during growth of BP504. Growth of BP504 was monitored by measuring the optical density nm for 24 h (0). At various time intervals, a sample of culture was taken and the whole cells were washed and assayed directly for ACT enzymatic activity (O). (B) ACT biological and enzymatic activities in urea extracts of BP504 peaks at 10 h of growth. Samples were taken as for panel A, and the washed cells were extracted with 6 M urea. Dialyzed extract supernatants were assayed for both biological (0) and enzymatic (C1) activities. at 650

results of these experiments are shown in Fig. 1. Evaluation of the amount of cell-associated ACT was based primarily on the enzymatic activity of the whole-cell samples. Initial production of ACT occurred at about 2 h postinoculation. Synthesis of cell-associated ACT continued during log-phase growth and peaked at 10 h postinoculation. The presence of cell-associated ACT gradually declined over the next 12 h to quantities less than 50% of peak enzymatic levels (Fig. 1A). Culture supernatant ACT levels reached a plateau at about 16 h postinoculation but were significantly lower than levels associated with cells at 10 h (data not shown). In separate experiments, cells were harvested at specific time points and urea extracted, and the extract was then tested for both ACT biological and enzymatic activities. Both activities peaked at 10 h (Fig. 1B). Purification of ACT from whole-cell urea extracts. Enzymatically and biologically active ACT had been shown previously to be recovered by one-step chromatography with calmodulin-Sepharose affinity chromatography (4, 5). Preparations obtained by this method were of variable purity. A single protein of 220 kDa could be consistently recovered by altering the loading conditions for the crude extract from 50 mM NaCl to 150 mM and including 2 mM CHAPS in the buffer. Preparations of affinity-purified ACT exhibiting the single band at 220 kDa were positive for FHA when screened by Western immunoblot with polyclonal affinity-purified ot-FHA. No reaction was observed when the blots were probed with affinity purified anti-PT (data not

shown). These data suggested that FHA was copurifying with ACT. FHA was removed from ACT by initially passing crude urea extracts of whole-cell BP504 over an HA column (24). In this step, ACT was bound to HA in 10 mM phosphate buffer (pH 8.0). ACT was eluted from the column by increasing the concentration of phosphate to 100 mM. Under these conditions, FHA remained bound to the HA, as confirmed by the absence of a reaction in blots of eluant fractions probed with affinity-purified a-FHA antibody (data not shown). The eluant fractions containing ACT activity were pooled and passed over calmodulin-Sepharose. The active fractions eluted from calmodulin-Sepharose contained extremely small quantities of protein but contained nearly 2,000 U of enzymatic activity (Fig. 2A). The protein contents of individual active fractions were too low to measure or visualize unless the fractions were pooled and lyophilized prior to protein assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The primary protein recovered was 220 kDa (Fig. 2B). A summary of the purification (Table 1) suggests that while the protein recovery was low, the specific enzymatic activity was approximately 17,000 U/mg! min, representing a 465-fold purification from crude urea extracts.

Purified 220 kDa ACT has biological activity. Biological activity of purified ACT was demonstrated by both CL inhibition in neutrophils and by the increase in intracellular cAMP in S49 lymphoma cells. The data in Fig. 3 were




TABLE 1. ACT purification profile



F lo. TP,,u









E 0 U,

Nvc 0.2

E 1000





' !




20 Fraction # 15



0 o


1 2



Step" C

194 e


20.7- ^ 16.3FIG. 2. (A) Calmodulin-Sepharose chromatogram of active fractions pooled from HA chromatography. *, A280; O, enzymatic activity. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lyophilized pool of fractions 25 to 31 from chromatogram depicted in panel A. Lanes: 1, molecular mass standards; 2, lyophilized pool (5 ,ug). Sizes on the left are in kilodaltons.

representative of the biological activity of purified ACT and showed that the eluant pool from calmodulin-Sepharose had greater ability to inhibit neutrophil CL than the crude cellular extract. The quantity of protein used to induce a 98% inhibition response in 2 x 106 neutrophils per ml was 8 ,ug. This quantity was substantially lower than the 50% inhibition dose previously shown (5). Quantities of ACT as small as 660 ng inhibited the CL response by 64% in 2 x 106 neutrophils per ml. Furthermore, significant increases in intracellular cAMP could be induced in S49 cells by using less than 1 p,g of purified ACT. As little as 2.5 ng of ACT induced 938 pmol of cAMP per 2 x 106 S49 cells. The biological activity of purified ACT was 6,250 U/mg/min and represents a 8,933fold purification from crude urea extracts (Table 1).

1 2 3




56.8 33.7 0.1

2,070 2,139 2,165

Sp Act/mg Biological"

Enzymaticb 36.5 63.5

0.7 0.5





1.0 1.7 465.3

" 1, Whole-cell crude extract; 2, HA eluant pool; 3, calmodulin-Sepharose eluant pool. b Micromoles of cAMP per minute. ' Nanomoles of cAMP per minute. " Calculated by using enzymatic specific activity.

Interestingly, the biological activity of purified ACT was extremely unstable at the small protein quantities recovered following affinity chromatography. The biological activities of purified preparations could be maintained by including extraneous proteins such as BSA (0.1 mg/ml) in the ureaeluted fractions. Paired pools from affinity chromatography assayed for increasing cAMP in S49 cells maintained their toxin activities better when BSA was included in the pool (data not shown). Isoelectric point. The isoelectric point of ACT was determined. A pool of several affinity-purified ACT preparations was focused in a Rotofor liquid isoelectric-focusing cell. Fractions recovered from the Rotofor with a pH of approximately 7.0 contained adenylate cyclase enzymatic activity

(Fig. 4). DISCUSSION In order to obtain the maximal amount of both biologically and enzymatically active ACT for purification, it was important to determine the point in the BP504 growth cycle at which maximal toxin was being produced. BP504 demonstrated a peak of both activities during mid-log-phase growth at 10 h. Previously, only one detailed investigation into the correlation between growth of B. pertussis and the production of enzymatically active ACT had been done (3). Four phase I strains, 3779 B, Tohama, 18323, and Maeno, were investigated, and the average optimal time of cell-associated ACT was about 15 h postinoculation. The levels of cellassociated ACT would then gradually decline during the 100









Urea Ext.

Flow Thru


Fraction FIG. 3. CL inhibition in human neutrophils by ACT. Neutrophils were treated with 50 ,ul of buffer (control), preparations of urea extract (12 ,ug of total protein), calmodulin flowthrough pool (15 p.g), or calmodulin eluant pool (8 ,ug). Neutrophil viability was routinely >95% after incubation with ACT preparations, as measured by trypan blue exclusion.

VOL. 58, 1990



8 Q















Fraction # FIG. 4. ACT enzyme activity recovered following isoelectric focusing. A pool of several affinity-purified ACT preparations was focused in 6 M urea in a Rotofor isoelectric-focusing cell. pH; -, enzymatic activity. Fractions with a pH of 7.0 contained ACT enzymatic activity. --,


remainder of the growth cycle. The earlier production of cell-associated ACT in BP504 in comparison with that in these other strains is likely the result of strain differences. By contrast, the levels of supernatant enzymatic ACT levels did not peak until some 30 to 40 h into the growth cycle (3). Presumably, this increase in supernatant activity was the result of leakage from lysed B. pertussis cells. These findings supported earlier results in which 80% of the enzymatic activity was determined to be cell associated (11). Differences in production of cell-associated ACT have also been demonstrated in TnS mutants arising from the parental strain BP338 (25). We performed growth experiments with the FHA-negative mutant strain BP353 as a possible solution to the FHA contamination and found a similar peak time of cell-associated ACT. The majority of enzymatic activity was located in the culture supernatant fraction. However, the level of enzymatic activity in the culture supernatant was only about 50% of BP504 cell-associated activity (data not shown). It has been suggested that a series of B. pertussis genes, homologous with the hemolysin transport genes in Escherichia coli, may be involved with the transport of ACT (8). Since FHA is also a surface-associated protein, it is possible that competition exists between all surface molecules for transport machinery. Consequently, in the mutant BP353, more sites may be available to ACT, which results in higher levels of toxin in the culture supernatant. It would be most interesting if the BP353 ACT from culture supernatant retains its biological activity. One question left unanswered by the growth curve experiments is the relationship of ACT production in liquid culture to the in vivo disease state. While no direct comparisons can be made, it is unlikely that the expression of ACT in vivo would follow the pattern seen in liquid culture. Factors such as increased concentrations of metabolites or depletion of specific nutrients undoubtedly contribute significantly to the regulation of ACT synthesis in vitro. Additionally, it seems probable that B. pertussis would require the presence of the toxin, whether cell associated or extracellular, for much greater periods in vivo to avoid possible destruction by phagocytic cells. Previously, the purification of ACT was described with a single calmodulin-Sepharose affinity chromatography step (4). A single peak, containing a major protein at 60 kDa and a minor protein at approximately 200 kDa, was eluted with EGTA and contained both biological and enzymatic activities. The levels of both these activities were low. In light of


the results described here, the 220-kDa protein, which contains both of the desired activities of ACT, was separated from the inactive 60-kDa protein by varying the loading conditions. Increasing the NaCl and detergent concentrations was sufficient to disrupt binding of the 60-kDa protein. This result suggests that the previous appearance and misidentification of the 60-kDa protein as the ACT was the result of nonspecific interactions of this protein with either the affinity resin or the 220-kDa ACT. Substantially greater amounts of purified 220-kDa ACT with both high enzymatic and biological specific activities were recovered by elution of the affinity resin with 6 M urea (Table 1). Apparently, ACT binds to the calmodulin-Sepharose too avidly to be dissociated by ethylene glycol-bis(paminoethyl ether)-N,N,N',N',-tetraacetic acid (EGTA). This possibility has been suggested previously (23). While EGTA is capable of blocking the stimulatory effects of calmodulin by preventing Ca2'-dependent association with ACT, this occurs only when EGTA is added prior to calmodulin (27). Thus, once the ACT-calmodulin interaction has taken place, EGTA is incapable of disrupting the association. A dissociation constant for ACT and calmodulin in the presence of EGTA has been reported to be 108 M, while the dissociation constant in the presence of Ca2+ was 1010 M (14). This could account for the greater activities recovered with 6 M urea for elution. Most surprising was the recovery of FHA with ACT following affinity chromatography. While no association between these two virulence determinants has been previously shown, it seems likely that the copurification is the result of hydrophobic interaction between the two proteins. Both FHA and ACT appear to have exposed regions of hydrophobicity. Each of these proteins avidly binds to the hydrophobic interaction resin phenyl-Sepharose (data not shown). The derived amino acid sequence for FHA demonstrates three regions of hydrophobicity, two of which are in areas believed to be involved with binding domains of FHA to eucaryotic cells (21). The cell association of ACT certainly implies a hydrophobic nature for this protein. Furthermore, penetration of ACT into eucaryotic cells may require direct interaction with the cell membrane (9). Fortunately, FHA can be easily removed from crude extracts by use of HA chromatography. An unfortunate consequence of the low protein yields of homogeneously purified ACT is the rapid degradation of its biological activity. Preparations of affinity-purified 220-kDa ACT, stored at low protein concentrations, have been visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have degraded into multiple lower-molecularmass proteins (data not shown). The biological activity of ACT can be stabilized by including proteins such as BSA at 0.1 mg/ml. B. pertussis produces maximal ACT in vitro during a period of active cell growth. In BP504, this time corresponds to 10 h; however, variability of peak toxin production is likely to exist among the different strains of B. pertussis currently being used for investigation of ACT. Similarly, the compartmental location of ACT may also differ among strains. While BP504 ACT activity is predominantly cell associated, this was not the case for the FHA-negative mutant BP353. The purified, native ACT is a protein with a molecular mass of 220 kDa and an apparent pl of 7.0. Biological activity is clearly associated with this molecular species. Although the protein appears to be produced in low quantities, its activities suggest that it is quite potent. ACT also exhibits physical characteristics which are consistent



with its cellular location as well as the nature of its penetration into target cells. The apparent nonspecific association with other proteins (e.g., FHA) and the need for some proteins to help stabilize activity (e.g., BSA) suggest that protein-protein interactions may also be important in vivo. This raises further questions as to whether synergistic roles exist between ACT and the other virulence determinants of B. pertussis. ACKNOWLEDGMENTS We thank Bill Cuevas for assistance with the Rotofor isoelectric focusing and Sue Waite for the cAMP quantitation assay protocol. Charles Sterling, Lynn Joens, Kenneth Ryan, and Lisa Steed for their critical review of the manuscript. This work was supported by Public Health Service grant RO1A122822 from the National Institutes of Health and a Flinn Foundation grant (044-100) to R.L.F. LITERATURE CITED 1. Blackshear, P. J. 1984. Systems for polyacrylamide gel electrophoresis. Methods Enzymol. 104C:237-255. 2. Confer, D. L., and J. W. Eaton. 1982. Phagocyte impotence caused by an invasive adenylate cyclase. Science 217:948-950. 3. Endoh, M., T. Takezawa, and Y. Nakase. 1980. Adenylate cyclase activity of Bordetella organisms. I. Its production in liquid medium. Microbiol. Immunol. 24:95-104. 4. Friedman, R. L. 1987. Bordetella pertussis adenylate cyclase: isolation and purification by calmodulin-sepharose 4B chromatography. Infect. Immun. 55:129-134. 5. Friedman, R. L., R. L. Fiederlein, L. Glasser, and J. N. Galgiani. 1987. Bordetella pertussis adenylate cyclase: effects of affinitypurified adenylate cyclase on human polymorphonuclear leukocyte functions. Infect. Immun. 55:135-140. 6. Gilman, A. G. 1970. A protein binding assay for adenosine 3':5'-cyclic monophosphate. Proc. Natl. Acad. Sci. USA 67: 305-312. 7. Glaser, P., D. Ladant, 0. 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. 8. Glaser, P., H. Sakamoto, J. Bellalou, A. Ullmann, and A. Danchin. 1988. Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis. EMBO J. 7:3997-4004. 9. 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. 10. 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.

INFECT. IMMUN. 11. Hewlett, E. L., M. A. Urban, C. R. Manclark, and J. Wolff. 1976. Extracytoplasmic adenylate cyclase of Bordetella pertussis. Proc. Natl. Acad. Sci. USA 73:1926-1930. 12. Hewlett, E. L., and J. Wolff. 1976. Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization. J. Bacteriol. 127:890-898. 13. Kessin, R. H., and J. Franke. 1986. Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms. J. Bacteriol. 166:290-296. 14. Ladant, D. 1988. Interaction of Bordetella pertussis adenylate cyclase with calmodulin: identification of two separate calmodulin-binding domains. J. Biol. Chem. 263:2612-2618. 15. Ladant, D., C. Brezin, J. M. Alonso, I. Crenon, and N. Guiso. 1986. Bordetella pertussis adenylate cyclase purification, characterization, and radioimmunoassay. J. Biol. Chem. 261:1626416269. 16. Morrissey, J. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310. 17. O'Farrell, P. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021. 18. Olson, L. C. 1975. Pertussis. Medicine (Baltimore) 54:427-469. 19. 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. 20. Raptis, A., L. Knipling, and J. Wolff. 1989. Dissociation of catalytic and invasive activities of Bordetella pertussis adenylate cyclase. Infect. Immun. 57:1725-1730. 21. Relman, D. A., M. Domenighini, E. Tuomanen, R. Rappuoli, and S. Falkow. 1989. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc. Natl. Acad. Sci. USA 86:2637-2641. 22. Rogel, A., Z. Farfel, S. Goldschmidt, J. Shiloach, and E. Hanski. 1988. Bordetella pertussis adenylate cyclase: identification of multiple forms of the enzyme by antibodies. J. Biol. Chem. 263:13310-13316. 23. Rogel, A., J. E. Schultz, R. M. Brownlie, J. G. Coote, R. Parton, and E. Hanski. 1989. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme. EMBO J. 8:2755-2760. 24. Sato, Y., J. L. Cowell, H. Sato, D. G. Burstyn, and C. R. Manclark. 1983. Separation and purification of the hemagglutinins from Bordetella pertussis. Infect. Immun. 41:313-320. 25. Weiss, A. A., E. L. Hewlett, G. A. Myers, and S. Falkow. 1983. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect. Immun. 42:33-41. 26. White, A. A. 1974. Separation and purification of cyclic nucleotides by alumina column chromatography. Methods Enzymol. 38C:41-46. 27. 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.

Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization.

Bordetella pertussis produces a number of virulence determinants which contribute to its pathogenicity. One factor, the adenylate cyclase toxin (ACT),...
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