International Bordetella pertussis assay standardization and harmonization meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, United States, 19-20 July 2007.

An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19-20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO). The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.

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.

Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis.

Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP. To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B. pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin. Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin. A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B. pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication. B. pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria. Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria. These data establish that delivery of this toxin by intact B. pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin.

Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis.

Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca(2+) for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn(2+) EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn(2+) with a K(D) of 0.05 to 0.35 microM and one or more classes of lower affinity sites. Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five beta-sheet helices within the repeat domain of the toxin upon binding Ca(2+) is required for cell intoxication.

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.

Adenylate cyclase toxin from Bordetella pertussis: current concepts and problems in the study of toxin functions.

Adenylate cyclase (AC) toxin produced by Bordella pertussis and other Bordella species is a virulence factor and protective antigen with novel properties and activities, which make it attractive as a prototype toxin for study of membrane insertion and delivery to the target cell interior. It is unique among RTX toxins in that it possesses enzymatic (adenylate cyclase) activity, as well as the capacity to create an ion-permeable pore in target cell membranes and lyse erythrocytes. The current issues in understanding AC toxin, which will be discussed here, include the role of acylation in its various activities and the relationship among those several toxin functions.

Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of Bordetella pertussis by human neutrophils.

A previous study showed that opsonization with human immune serum could either promote or antagonize phagocytosis of Bordetella pertussis by human neutrophils depending on whether the bacteria expressed adenylate cyclase toxin. Opsonization of the wild-type strain inhibited phagocytosis relative to unopsonized controls. In contrast, mutants lacking adenylate cyclase toxin were efficiently phagocytosed when opsonized with human immune serum. In this study, we examined opsonization in the presence or absence of monoclonal antibodies to adenylate cyclase toxin. Addition of neutralizing monoclonal antibodies to adenylate cyclase toxin converted a serum that previously inhibited both attachment and phagocytosis of the wild-type strain to one that increased both attachment and phagocytosis compared to the no-serum control. Monoclonal antibodies that recognize the adenylate cyclase toxin but fail to neutralize activity were without effect. These results suggest that adenylate cyclase toxin inhibits both Fc receptor-mediated attachment and phagocytosis of B. pertussis by neutrophils.

Escherichia coli alpha-hemolysin (HlyA) is heterogeneously acylated in vivo with 14-, 15-, and 17-carbon fatty acids.

alpha-Hemolysin (HlyA) is a secreted protein virulence factor observed in certain uropathogenic strains of Escherichia coli. The active, mature form of HlyA is produced by posttranslational modification of the protoxin that is mediated by acyl carrier protein and an acyltransferase, HlyC. We have now shown using mass spectrometry that these modifications, when observed in protein isolated in vivo, consist of acylation at the epsilon-amino groups of two internal lysine residues, at positions 564 and 690, with saturated 14- (68%), 15- (26%), and 17- (6%) carbon amide-linked side chains. Thus, HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures, and the substrate specificity of the HlyC acyltransferase appears to differ from that of the closely related CyaC acyltransferase expressed by Bordetella pertussis.

Characterization of binding of adenylate cyclase toxin to target cells by flow cytometry.

Adenylate cyclase (AC) toxin from Bordetella pertussis intoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K(+) and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K(+) efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 micrograms/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37 degrees C; however, the total binding at 0 degrees C is less than that at 37 degrees C. In human erythrocytes, AC toxin does not cause an increase in K(+) efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K(+) efflux, and hemolysis produced by AC toxin.

A commentary on the pathogenesis of pertussis.

In recent years a great deal of information has been generated on the virulence factors produced by Bordetella pertussis, the regulation of their expression, and their molecular mechanisms of action. There are numerous studies of Bordetella virulence factors and strains of B. pertussis in which the genes for some of these components have been mutated or deleted. In addition, several acellular vaccines composed of these virulence factors have been developed, tested, and licensed for use in the prevention of pertussis. Nevertheless, there exists little information specifically on the pathogenesis of the disease process caused by B. pertussis in humans, and such data are necessary for adequate understanding and treatment of this novel infectious disease.

Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase (AC) toxin from Bordetella pertussis is a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.

Pertussis: current concepts of pathogenesis and prevention.

The selection of acellular vaccine antigens relies on current concepts of pertussis pathogenesis. Animal model data provide evidence that certain products of Bordetella pertussis, which include the putative adhesins filamentous hemagglutinin, pertactin and fimbriae, and pertussis toxin could serve as protective antigens and are available in sufficient quantities of purified material to be considered appropriate candidates for vaccine inclusion. In clinical studies vaccines containing three, four or five components were more effective at preventing pertussis than vaccines containing only inactivated pertussis toxin and filamentous hemagglutinin. These data suggest that pertactin may make a contribution to the protection elicited by an acellular product, but information does not allow evaluation of a possible incremental contribution from fimbriae. Serologic studies of patients in the clinical efficacy trials of the acellular pertussis vaccines did not yield a correlation between antibody levels and protection against pertussis, which suggests that relationships or mechanisms involved in the protective activities of these acellular vaccines are not yet understood. Therefore other mechanisms of immunity (i.e. cellular immunity) may be involved in vaccine-elicited immunity. Increasing understanding of the likely mechanisms of pertussis pathogenesis will provide insights into potential therapies for patients infected with B. pertussis. The mechanisms of vaccine-induced immunity remain elusive and determination of whether these products are working as initially predicted will require further study.

Role of pertussis toxin A subunit in neutrophil migration and vascular permeability.

The anti-inflammatory activity of pertussis toxin (Ptx) was compared to that of a noncatalytic mutant of pertussis toxin (9K/129G; Ptxm), which contains two amino acid substitutions in the A protomer, by using a rat model of inflammation. The toxins were administered intravenously 1 h prior to the injection of inflammatory stimuli. Ptx, but not Ptxm, inhibited neutrophil migration into peritoneal cavities in response to formyl-methionyl-leucyl-phenylalanine and lipopolysaccharide. The inhibitory effect of Ptx on neutrophil migration could not be explained by the ability of the toxin to induce leukopenia or neutropenia. The increase in skin vascular permeability induced by leukotriene B4, a powerful neutrophil chemotactic agent, was also inhibited only by Ptx. On the other hand, the increase in skin vascular permeability induced by histamine was potentiated by both toxins. These data show that Ptx inhibits neutrophil-mediated inflammation in vivo and that this effect is dependent on the ADP-ribosyltransferase activity of the A protomer.

Preparation and composition of acellular pertussis vaccines. Consideration of potential effects on vaccine efficacy.

An evaluation of possible relationships between the preparation and content of acellular pertussis vaccines and their efficacy reveals that, in most instances, there is much more extensive variability in content and mode of preparation of these products than in their clinical efficacies. In general, there is a positive correlation of antigen content with immunogenicity. However, there was an apparent difference in the dose-response relationships between chemically and genetically toxoided vaccines which revealed a greater immunogenicity of the genetically inactivated material. Overall, however, the immunigenicity data (IgG serology) do not correlate well with protective efficacy and it is apparent that additional information is necessary to elucidate a specific role for any aspect of vaccine content or preparation in vaccine efficacy.

Adenylate cyclase in striatal cholinergic interneurons regulates acetylcholine release.

Fractional [3H]ACH efflux from dissociated rat striata tested whether tonic inhibition prevents stimulation of acetylcholine (ACH) release by adenylate cyclase. Forskolin stimulated release from the dissociated cells (threshold at 300 nM; EC50 > or = 1 MicroM). Release was also stimulated by 3-isobutyl-1-methylxanthine and was additive with forskolin. The 1,9-dideoxy forskolin analog that lacks cyclase-stimulating activity was ineffective. Thus, stimulation of adenylate cyclase within striatal cholinergic interneurons increases ACH secretion but is tonically inhibited by endogenous striatal transmitters. Disinhibition of the excitatory cyclase by denervation of striatal cholinergic interneurons in situ could contribute to supersensitivity without receptor upregulation.

ATP shortens atrial action potential duration in the dog: role of adenosine, the vagus nerve, and G protein.

The mechanism by which adenosine 5'-triphosphate (ATP) shortens atrial action potential duration was studied in a canine model in vivo. Previous studies have indicated that the negative chronotropic and dromotropic actions of ATP in the canine heart are mediated by a vagal reflex and by adenosine. However, the mechanism of ATP's action on atrial action potential duration remains unknown. The effects of ATP on endocardial monophasic action potential were determined under baseline conditions (control) and after left cervical vagotomy followed by right vagus nerve afferent blockade with capsaicin (1% in olive oil), and subsequent bilateral cervical vagotomy plus propranolol (1.0 mg/kg, i.v.). In addition, the effects of ATP and adenosine were determined 48 h following the administration of pertussis toxin (PTX, 30 micrograms/kg, i.v.). PTX intoxication was verified by monitoring plasma levels of insulin during glucose tolerance tests. ATP (4 and 6 mumol/kg, rapid bolus into right atrium) markedly shortened right atrial action potential duration at 50% repolarization (APD50) from 101 +/- 8 to 22 +/- 6 and from 111 +/- 8 to 14 +/- 2 ms, respectively. Adenosine (equimolar doses given in an identical mode) had a smaller effect, i.e., APD50 of 106 +/- 8 and 109 +/- 6 was shortened to 77 +/- 12 and 76 +/- 12 ms, respectively. Left cervical vagotomy slightly reduced the effect of ATP but not that of adenosine. Blockade of right vagal C fiber afferent traffic using local application of capsaicin to the right cervical vagosympathetic trunk markedly attenuated the effect of ATP, but not that of adenosine. Autonomic blockade (i.e., bilateral cervical vagotomy and propranolol) markedly attenuated the effect of ATP, but not of adenosine; for example, the effect of ATP (6 mumol/kg) was reduced from 86 +/- 2% shortening of APD50 to 24 +/- 5% (p < 0.05), while that of adenosine was 32 +/- 8 and 20 +/- 4% (ns) before and after autonomic blockade, respectively. Treatment with PTX completely abolished the effect of both ATP and adenosine on atrial action potential duration. These data indicate that (i) the effect of ATP on the canine atrial action potential duration is mediated to a large extent by a vagal reflex triggered by the nucleotide and to a lesser extent by adenosine, the product of ATP's enzymatic degradation,(ii) the afferent traffic of this reflex travels mainly via the right vagal C fibers, and (iii) the effects of both vagal and the adenosine components are mediated by PTX-sensitive guanine nucleotide binding proteins (G proteins) coupled to the muscarinic cholinergic receptors and A1 adenosine receptors, respectively.

Hemolytic, but not cell-invasive activity, of adenylate cyclase toxin is selectively affected by differential fatty-acylation in Escherichia coli.

Adenylate cyclase toxin from Bordetella pertussis requires posttranslational acylation of lysine 983 for the ability to deliver its catalytic domain to the target cell interior and produce cyclic adenosine monophosphate (cell-invasive activity) and to form transmembrane channels (hemolytic activity). When the toxin is expressed in Escherichia coli, it has reduced hemolytic activity, but comparable cell-invasive activity to that of adenylate cyclase toxin from B. pertussis. In contrast to the native protein from B. pertussis, which is exclusively palmitoylated, recombinant toxin from E. coli is acylated at lysine 983 with about 87% palmitoylated and the remainder myristoylated. Furthermore, the recombinant toxin contains an additional palmitoylation on approximately two-thirds of the lysines at position 860. These observations suggest that the site and nature of posttranslational fatty-acylation can be dictated by the bacterial host used for expression and can have a significant, but selective, effect on protein function.

Membrane depolarization prevents cell invasion by Bordetella pertussis adenylate cyclase toxin.

Adenylate cyclase toxin from Bordetella pertussis is a 177-kDa calmodulin-activated enzyme that has the ability to enter eukaryotic cells and convert endogenous ATP into cAMP. Little is known, however, about the mechanism of cell entry. We now demonstrate that intoxication of cardiac myocytes by adenylate cyclase toxin is driven and controlled by the electrical potential across the plasma membrane. The steepness of the voltage dependence of intoxication is comparable with that previously observed for the activation of K+ and Na+ channels of excitable membranes. The voltage-sensitive process is downstream from toxin binding to the cell surface and appears to correspond to the translocation of the catalytic domain across the membrane.