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Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartaterich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca2+, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C-terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.

Adenylate cyclase (AC) toxin from Bordetella pertussis penetrates eukaryotic cells and upon activation by calmodulin generates unregulated levels of intracellular cAMP. The process of toxin penetration into sheep erythrocytes was resolved into three consecutive steps including insertion, translocation, and intracellular cleavage. Insertion of the toxin into the cell membrane occurred over a wide temperature range (4-36 degrees C). In contrast, translocation of the toxin, i.e. transfer of the NH2-terminal catalytically active fragment across the membrane, occurred only above 20 degrees C and was highly temperature-dependent. While a single exposure of the toxin to Ca2+ was sufficient for its insertion into the plasma membrane, toxin translocation required exogenous Ca2+ at mM concentrations. Translocation was not affected by pretreatment of cells with trypsin, N-ethylmaleimide, and sodium carbonate at alkaline pH. The NH2-terminal fragment of the toxin was cleaved in the cell releasing the 45-kDa active AC into the cytosol. The cleavage was blocked by treatment of cells with N-ethylmaleimide. It is hypothesized that the COOH-terminal portion of the toxin creates in the membrane a channel through which the NH2-terminal fragment is translocated.

Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms. Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP. Recombination studies in transposon Tn5-insertion mutants of B. pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity. Here, we demonstrate that only the toxic form of B. pertussis AC possesses hemolytic activity. Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B. pertussis AC. cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity. Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation. Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis. However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP. Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis. These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.

Bordetella pertussis produces extracytoplasmic adenylate cyclase toxin (AC toxin) which penetrates target cells and, upon activation by host calmodulin, generates high levels of intracellular cyclic AMP (cAMP). As a result, bactericidal functions of immune effector cells are impaired. Since a considerable amount of AC toxin is associated with the bacterium, it was proposed that the toxin may be delivered by direct interaction of the organism with the target cells (E. L. Hewlett, M. C. Gray, and R. D. Pearson, Clin. Res. 35:477A, 1987). Incubation of CHO cells with intact B. pertussis led to formation of intracellular cAMP at levels comparable to those produced in CHO cells by equivalent activities of isolated AC toxin. cAMP accumulation induced by the whole bacteria appeared after a lag of 40 to 60 min and reached high levels within 2 to 3 h, whereas adherence of the bacteria proceeded rapidly and reached a maximal level within 80 min. Sera of pertussis patients completely blocked cAMP accumulation induced by the whole bacteria without having a major effect on either bacterial adherence or cAMP production by the AC toxin. Cytochalasins B and D, inhibitors of bacterial invasion, abrogated the cAMP response to the whole bacteria but not the response to the AC toxin. These agents did not affect bacterial adherence. Transmission electron micrographs revealed that B. pertussis, within the time course of cAMP induction, invaded CHO cells. We suggest that cAMP induction by B. pertussis is caused by the entry of the whole bacteria into CHO cells rather than by delivery of AC toxin during bacterial adherence. This route of cell intoxication may be relevant to the pathogenesis of whooping cough.

Bordetella pertussis produces several potential virulence factors. One of these is an adenylate cyclase which penetrates eukaryotic cells, is activated by calmodulin and generates high levels of intracellular cAMP. We have found that pertussis infection in man leads to production of high titres (2000-8000) of anti-B. pertussis adenylate cyclase antibodies. Such antibodies also are produced after pertussis vaccination. They persist into adulthood, cross the placenta and disappear a few months after birth. The anti-adenylate cyclase antibodies found in human serum during pertussis infection do not neutralise the catalytic and penetrative activities of the enzyme.

The existence of an invasive adenylate cyclase in dialyzed urea extracts of the bacterium Bordetella pertussis has been suggested recently. Gel filtration of B. pertussis dialyzed urea extract shows that the invasive enzyme constitutes only a small portion of the total adenylate cyclase activity found in the extract. Its size is different than the size of the two peaks of adenylate cyclase activity identified in the extract. Ca2+ is absolutely required for the penetration of the invasive enzyme, it also controls the rate of intracellular cAMP accumulation in human lymphocytes exposed to dialyzed extract. These characteristics may be attributed to the increase in the size of the invasive enzyme as found by gel filtration chromatography of the extract in the absence of Ca2+. Removal of nonpenetrating adenylate cyclase that adheres to lymphocytes permits a direct assay of the intracellular enzyme. The time course of intracellular accumulation of adenylate cyclase activity is similar to the time course of intracellular accumulation of cAMP, suggesting that the invasive enzyme is rapidly deactivated, but not degraded, since it can be detected upon cell disruption. No appreciable amount of the enzyme is introduced when cells are incubated with extract at 4 degrees C for 120 min, then washed and incubated further at 37 degrees C. Concanavalin A inhibits cAMP accumulation irrespective of the time of its addition, and EGTA prevents penetration of the invasive enzyme even if added 20 min after addition of extract. These findings are different from those observed in other bacterial toxins thought to be internalized by receptor-mediated endocytosis. However, the cellular penetration of B. pertussis adenylate cyclase is cell-selective. It does not occur in human erythrocytes. In addition to human lymphocytes, S49 cyc- murine lymphoma, turkey erythrocytes, and rat oocytes accumulate cAMP in response to B. pertussis extract.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.

Bordetella pertussis produces an adenylate cyclase which is a toxin. The enzyme penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP; as a result bactericidal functions of immune effector cells are considerably impaired. The toxin is composed of a single polypeptide that possesses both the catalytic and the toxic functions. It penetrates the host cell directly from the plasma membrane and is concomitantly inactivated by a proteolytic degradation.

Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.

Bordetella pertussis produces a calmodulin-dependent adenylate cyclase (AC) which acts as a toxin capable of penetrating eukaryotic cells and generating high levels of intracellular cyclic AMP. Transfer of target cells into B. pertussis AC-free medium leads to a rapid decay in the intracellular AC activity, implying that the invasive enzyme is unstable in the host cytoplasm. We report here that treatment of human lymphocytes with a glycolysis inhibitor and an uncoupler of oxidative phosphorylation completely blocked the intracellular inactivation of B. pertussis AC. Lymphocyte lysates inactivated all forms of B. pertussis AC in the presence of exogenous ATP. This inactivation was associated with degradation of an 125I-labelled 200 kDa form of B. pertussis AC. It appears that ATP is required for the proteolytic pathway, but not as an energy source, since non-hydrolysable ATP analogues supported inactivation and complete degradation of the enzyme. The possibility that binding of ATP to B. pertussis AC renders it susceptible to degradation by the host cell protease is discussed.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. The spontaneous process was studied in pathogenic B. pertussis strains Tohama, 165 and 18323 by isolating spontaneous variants, selected for their ability to grow on synthetic and semi-synthetic solid media. In strains Tohama and 165, the frequency of variants able to grow on synthetic and semi-synthetic media was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried pertussis toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin was related quantitatively to the other traits. These results suggest that phase variation in B. pertussis is a non random process, involving ordered disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. Since all the variant strains were phenotypically stable upon further passaging in vitro, they represent the stable, final outcome of the variation process which may have occurred in "Phase I" colonies. In contrast, 300 variants of B. pertussis 18323, which were able to grow on selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral virulence.

Two forms of Bordetella pertussis adenylate cyclase of 200 and 47 kDa have been purified from dialyzed urea extract of the bacteria to specific activities of 466 and 1685 mumol.min-1.mg-1, respectively. Both forms are activated 50-200-fold by calmodulin. The half-maximum concentration required for the activation of the 200-kDa catalyst is 5.4.10(-9) M, whereas the one required for activation of the 47-kDa catalyst is 1.8.10(-10) M. Polyclonal antibodies raised against the 47-kDa catalyst specifically recognize both forms of the enzyme in purified state as well as in bacterial extracts on immunoblots. The antibody inhibits at similar titer adenylate cyclase activity of the purified forms as well as the activity present in dialyzed urea extract of the bacteria. It also prevents the penetration of the invasive B. pertussis adenylate cyclase into human lymphocytes. The inhibition induced by the antisera is specific to B. pertussis enzyme, since both calmodulin-dependent brain and sperm adenylate cyclase are not affected by the antibody.

A recombinant plasmid, pRMB1, identified from a gene library of B. pertussis, restored adenylate cyclase (AC) and haemolysin (HLY) activities to B. pertussis BP348 (a Tn5-insertion mutant deficient in both these activities). B. pertussis BP348 was considerably less virulent than wild type strains of B. pertussis when 3-week-old mice were challenged intranasally; possession of pRMB1 restored virulence. Neither AC nor HLY activities were expressed in E. coli harbouring pRMB1. However, expression of calmodulin-responsive AC was obtained in E. coli when restriction fragments of pRMB1 were subcloned into other vectors; expression depended on the lac and tac promoters of these vectors. The enzyme was not readily solubilized from urea extracts of E. coli and required sonication for efficient release. One plasmid conferred a specific AC enzymic activity to E. coli which was greater than that for wild type B. pertussis strains. Unlike extracts of B. pertussis, extracts from E. coli expressing enzymic AC activity, did not elevate cAMP levels in S49 lymphoma cells. A second plasmid, pRMB2, was identified from the gene library, which contained a trans-acting regulatory determinant required for expression of AC, HLY and other virulence-associated factors in B. pertussis.

Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5'-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium.

The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5'-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane.