Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells

Adenylate cyclase (AC) toxins produced by Bacillus anthracis and Bordetella pertussis were compared for their ability to interact with and intoxicate Chinese hamster ovary cells. At 30 degrees C, anthrax AC toxin exhibited a lag of 10 min for measurable cAMP accumulation that was not seen with pertussis AC toxin. This finding is consistent with previous data showing inhibition of anthrax AC toxin but not pertussis AC toxin entry by inhibitors of receptor-mediated endocytosis (Gordon, V. M., Leppla, S. H., and Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069). Treatment of target Chinese hamster ovary cells with trypsin or cycloheximide reduced anthrax AC toxin-induced cAMP accumulation by greater than 90%, but was without effect on pertussis AC toxin. In contrast, incubation of the AC toxins with gangliosides prior to addition to target cells inhibited cAMP accumulation by pertussis AC toxin, but not anthrax AC toxin. To evaluate the role of lipids in the interaction of pertussis AC toxin with membranes, multicompartmental liposomes were loaded with a fluorescent marker and exposed to toxin. Pertussis AC toxin elicited marker release in a time- and concentration-dependent manner and required a minimal calcium concentration of 0.2 mM. These data demonstrate that the requirements for intoxication by the AC toxins from B. anthracis and B. pertussis are fundamentally different and provide a perspective for new approaches to study the entry processes.     

Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.

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.     

Adenylate cyclase toxin from Bordetella pertussis. Identification and purification of the holotoxin molecule

Bordetella pertussis adenylate cyclase (AC) toxin is a calmodulin-activated adenylate cyclase enzyme which has the capacity to enter eukaryotic target cells and catalyze the conversion of endogenous ATP into cyclic AMP. In this work, the AC holotoxin molecule is identified and isolated. It is a single polypeptide of apparent 216 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies which immunoprecipitate AC activity from extracts of wild type B. pertussis (BP338) react with this 216-kDa band on Western blots, and it is absent from a transposon Tn5 mutant (BP348) specifically lacking AC toxin. Isolation of the 216-kDa protein to greater than 85% purity by hydrophobic chromatography, preparative sucrose gradient centrifugation, and affinity chromatography using either calmodulin-Sepharose or monoclonal antibody coupled to Sepharose 4B yields stepwise increases in AC toxin potency, to a maximum of 88.3 mumol of cAMP/mg of target cell protein/mg of toxin. Electroelution of the 216-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis yields a preparation with both AC enzyme and toxin activities. These data indicate that this protein represents the AC holotoxin molecule.     

Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX ( r epeats in t o x in) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with non-overlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.     

Translocation-specific conformation of adenylate cyclase toxin from Bordetella pertussis inhibits toxin-mediated hemolysis

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, DeltaAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to DeltaAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, DeltaN489 (missing amino acids 6 to 489) and DeltaN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while DeltaN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.     

Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone.     

Modification of biological responses to interleukin-1 by agents that perturb signal transduction pathways.

In this study we have examined the effect of agents known to perturb certain signal transduction pathways on the biological responses of target cells to stimulation with interleukin-1 (IL-1). In the murine thymoma cell line EL4, IL-1 stimulation results in the secretion of interleukin-2 (IL-2), which was subsequently measured by proliferation of an IL-2-dependent cell line. Agents that elevated intracellular cAMP blocked or partially blocked IL-1 induction of IL-2 secretion, whereas agents that activated protein kinase C (PKC) resulted in a synergistic enhancement. Both pertussis and cholera toxins also inhibited IL-1-induced IL-2 secretion, although probably by acting at different levels. IL-1 simulation of human and murine fibroblasts resulted in release of prostaglandin E2. This response was inhibitable by pertussis toxin but not by cholera toxin, whereas co-stimulation of the fibroblasts with IL-1 and phorbol ester resulted in a synergistic response. Murine fibroblasts could also be stimulated to proliferate by IL-1, and this response was also inhibitable by pertussis toxin. These findings are consistent with coupling of the IL-1 receptor to a signalling pathway via a pertussis toxin substrate.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Interaction of Bordetella pertussis virulence components with neutrophils: effect on chemiluminescence induced by a chemotactic peptide and by intact bacteria

The effect of secreted virulence components of Bordetella pertussis on chemiluminescence (CL) of rabbit peritoneal neutrophils was determined with the chemotactic peptide N'-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) or intact B. pertussis as the stimulus. Pertussis toxin (PT) inhibited the response to fMLP in a dose-dependent manner, although only after the neutrophils had been exposed to the toxin for greater than 15 min. Both filamentous haemagglutinin (FHA) and lipopolysaccharide (LPS) markedly enhanced the CL response to fMLP after greater than or equal to 15 min incubation with the neutrophils. Similar effects to those of B. pertussis LPS were also seen with smooth and rough LPS from Salmonella minnesota. With the lowest dose of each component which elicited a maximal effect on CL, the inhibitory effect of PT overrode the enhancing effect of FHA and B. pertussis LPS. Pre-incubation of neutrophils with PT, FHA or B. pertussis LPS caused a slight reduction in the subsequent CL response to virulent B. pertussis Tohama. Virulent (phase I, or X-mode) organisms of B. pertussis 18334 and B. pertussis Tohama induced greater neutrophil CL than their avirulent (C-mode) derivatives. There appeared to be an inverse correlation between bacterial hydrophilicity and the ability to induce neutrophil CL: X-mode bacteria were significantly less hydrophilic than C-mode organisms. Three mutants, the adenylate cyclase (AC)- and haemolysin (HLY)-deficient B. pertussis BP348, the FHA-deficient B. pertussis BP353, and the PT-deficient B. pertussis BP357, generated similar levels of CL and had similar hydrophilicity values. The hydrophilicity value of the avirulent mutant B. pertussis BP347 (deficient in AC, HLY, FHA and PT) and the CL induced by this strain were similar to those of B. pertussis C-mode organisms. Thus, the interaction of B. pertussis with neutrophils appears to be complex, reflecting both the alteration of leucocyte function by secreted virulence components of the organism and, in the absence of opsonins, the surface properties of the bacterium.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

Pertussis toxin blocks both cyclic AMP-mediated and cyclic AMP-independent actions of somatostatin. Evidence for coupling of Ni to decreases in intracellular free calcium

The neuropeptide somatostatin inhibits hormone release from GH4C1 pituitary cells via two mechanisms: inhibition of stimulated adenylate cyclase and a cAMP-independent process. To determine whether both mechanisms involve the guanyl nucleotide-binding protein Ni, we used pertussis toxin, which ADP-ribosylates Ni and thereby blocks its function. Pertussis toxin treatment of GH4C1 cells blocked somatostatin inhibition of both vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation and prolactin secretion. In membranes prepared from toxin-treated cells, somatostatin inhibition of VIP-stimulated adenylate cyclase activity was reduced and 125I-Tyr1-somatostatin binding was decreased more than 95%. In contrast, pertussis toxin did not affect the biological actions or the membrane binding of thyrotropin-releasing hormone. These results indicate that ADP-ribosylated Ni cannot interact with occupied somatostatin receptors and that somatostatin inhibits VIP-stimulated adenylate cyclase via Ni. To investigate somatostatin's cAMP-independent mechanism, we used depolarizing concentrations of K+ to stimulate prolactin release without altering intracellular cAMP levels. Measurement of Quin-2 fluorescence showed that 11 mM K+ increased intracellular [Ca2+] within 5 s. Somatostatin caused an immediate, but transient, decrease in both basal and K+-elevated [Ca2+]. Consistent with these findings, somatostatin inhibited K+-stimulated prolactin release, also without affecting intracellular cAMP concentrations. Pertussis toxin blocked the somatostatin-induced reduction of [Ca2+]. Furthermore, the toxin antagonized somatostatin inhibition of K+-stimulated and VIP-stimulated secretion with the same potency (ED50 = 0.3 ng/ml). These results indicate that pertussis toxin acts at a common site to prevent somatostatin inhibition of both Ca2+- and cAMP-stimulated hormone release. Thus, Ni appears to be required for somatostatin to decrease both cAMP production and [Ca2+] and to inhibit the actions of secretagogues using either of these intracellular messengers.     

Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity

Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.     

A study of the ability of Bordetella pertussis toxin to induce the formation of B-suppressors

Mouse spleen cells not adhering to the plastic surface and B-cells isolated from them were treated with B. pertussis toxin in vitro, washed and injected into recipients (allogeneic, syngeneic, intact or lethally irradiated) whose immune response to sheep red blood cells was then evaluated by Jerne's method. Treatment with B. pertussis toxin was shown to induce the development of immunosuppressive activity in intact spleen cells and in B-cells, to abolish the activity of memory B-cells and to enhance the suppressor activity of autoimmune mice. Supernatants obtained after autoimmune mice. Supernatants obtained after the 18- to 24-hour cultivation of spleen cells, previously treated with B. pertussis toxin for 60 minutes, suppressed the reaction of blast transformation of spleen cells to Con A and lipopolysaccharide and induced the appearance of immunosuppressive activity in intact spleen cells. The suppressing effect of the cells studied in this investigation may be linked with the ability of B. pertussis cells to stimulate the synthesis of cAMP, prostaglandins E and/or suppressor factors.     

Pancreastatin: a novel peptide inhibitor of parietal cell signal transduction

The direct inhibition of secretion by pancreastatin was investigated in rabbit isolated parietal cells. Pancreastatin exerted no influence on basal aminopyrine uptake. Pancreastatin inhibited histamine stimulated aminopyrine uptake through a decrease in intracellular cAMP. Pancreastatin inhibition of histamine stimulated uptake was blocked in the presence of pertussis toxin. Pancreastatin also inhibited the carbachol stimulated increase in aminopyrine accumulation. However, the effects of pancreastatin on carbachol stimulation were not reversed by pertussis toxin. Pancreastatin did not alter the carbachol induced increase in cytosolic free calcium. Thus, pancreastatin appears to inhibit parietal cell signal transduction at multiple points along the second messenger pathways.     

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

Distinct steps in the penetration of adenylate cyclase toxin of Bordetella pertussis into sheep erythrocytes. Translocation of the toxin across the membrane.

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.     

Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.     

The ins and outs of pertussis toxin

Pertussis toxin, produced and secreted by the whooping cough agent Bordetella pertussis, is one of the most complex soluble bacterial proteins. It is actively secreted through the B. pertussis cell envelope by the Ptl secretion system, a member of the widespread type IV secretion systems. The toxin is composed of five subunits (named S1 to S5 according to their decreasing molecular weights) arranged in an A-B structure. The A protomer is composed of the enzymatically active S1 subunit, which catalyzes ADP-ribosylation of the α subunit of trimeric G proteins, thereby disturbing the metabolic functions of the target cells, leading to a variety of biological activities. The B oligomer is composed of 1S2:1S3:2S4:1S5 and is responsible for binding of the toxin to the target cell receptors and for intracellular trafficking via receptor-mediated endocytosis and retrograde transport. The toxin is one of the most important virulence factors of B. pertussis and is a component of all current vaccines against whooping cough.