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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.

CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chinese hamster ovary (CHO) cells respond to pertussis toxin (PT) with a novel clustering pattern, which is dependent on biologically active PT. Since its description in 1983, this cellular response has been refined and used extensively for detection and quantification of PT activity, as well as anti-PT antibodies. There are limitations, however, in the use of this phenomenon as originally described. They are: (1) a subjective, observer-dependent scoring system; (2) the requirement for 16-24 h incubation in order for the response to be clearly detectable; and (3) apparent interference from non-toxin materials. To overcome these limitations, a number of alternative in vitro assays for PT, using CHO cells or other cell types, have been developed and are described elsewhere in this publication. In addressing the challenges associated with the CHO cell assay, we discovered that changes in the electrical impedance-based "normalized cell index" of PT-treated CHO cells obtained with the ACEA xCELLigence instrument enable objective detection/quantification of the PT-induced effect in as little as 3-4 h. To the best of our knowledge, the molecular basis for this intriguing response remains unknown. We present here electron microscopic (EM) images of control and PT-treated cells, which suggest some potential molecular mechanisms.

Bordetella pertussis is the causative agent of whooping cough, a serious respiratory illness affecting children and adults, associated with prolonged cough and potential mortality. Whooping cough has reemerged in recent years, emphasizing a need for increased knowledge of basic mechanisms of B. pertussis growth and pathogenicity. While previous studies have provided insight into in vitro gene essentiality of this organism, very little is known about in vivo gene essentiality, a critical gap in knowledge, since B. pertussis has no previously identified environmental reservoir and is isolated from human respiratory tract samples. We hypothesize that the metabolic capabilities of B. pertussis are especially tailored to the respiratory tract and that many of the genes involved in B. pertussis metabolism would be required to establish infection in vivo In this study, we generated a diverse library of transposon mutants and then used it to probe gene essentiality in vivo in a murine model of infection. Using the CON-ARTIST pipeline, 117 genes were identified as conditionally essential at 1 day postinfection, and 169 genes were identified as conditionally essential at 3 days postinfection. Most of the identified genes were associated with metabolism, and we utilized two existing genome-scale metabolic network reconstructions to probe the effects of individual essential genes on biomass synthesis. This analysis suggested a critical role for glucose metabolism and lipooligosaccharide biosynthesis in vivo This is the first genome-wide evaluation of in vivo gene essentiality in B. pertussis and provides tools for future exploration. IMPORTANCE Our study describes the first in vivo transposon sequencing (Tn-seq) analysis of B. pertussis and identifies genes predicted to be essential for in vivo growth in a murine model of intranasal infection, generating key resources for future investigations into B. pertussis pathogenesis and vaccine design., (Copyright © 2019 Gonyar et al.)

Bordetella bronchiseptica encodes and expresses a flagellar apparatus. In contrast, Bordetella pertussis , the causative agent of whooping cough, has historically been described as a nonmotile and nonflagellated organism. The previous statements that B. pertussis was a nonmotile organism were consistent with a stop codon located in the flagellar biosynthesis gene, flhA , discovered when the B. pertussis Tohama I genome was sequenced and analyzed by Parkhill et al. in 2003 (J. Parkhill, M. Sebaihia, A. Preston, L. D. Murphy, et al., Nat Genet, 35:32-40, 2003, https://doi.org/10.1038/ng1227). The stop codon has subsequently been found in all annotated genomes. Parkhill et al. also showed, however, that B. pertussis contains all genetic material required for flagellar synthesis and function. We and others have determined by various transcriptomic analyses that these flagellar genes are differentially regulated under a variety of B. pertussis growth conditions. In light of these data, we tested for B. pertussis motility and found that both laboratory-adapted strains and clinical isolates can be motile. Upon isolation of motile B. pertussis , we discovered flagellum-like structures on the surface of the bacteria. B. pertussis motility appears to occur primarily in the Bvg(-) phase, consistent with regulation present in B. bronchiseptica Motility can also be induced by the presence of fetal bovine serum. These observations demonstrate that B. pertussis can express flagellum-like structures, and although it remains to be determined if B. pertussis expresses flagella during infection or if motility and/or flagella play roles during the cycle of infection and transmission, it is clear that these data warrant further investigation. IMPORTANCE This report provides evidence for motility and expression of flagella by B. pertussis , a bacterium that has been reported as nonmotile since it was first isolated and studied. As with B. bronchiseptica , B. pertussis cells can express and assemble a flagellum-like structure on their surface, which in other organisms has been implicated in several important processes that occur in vivo The discovery that B. pertussis is motile raises many questions, including those regarding the mechanisms of regulation for flagellar gene and protein expression and, importantly, the role of flagella during infection. This novel observation provides a foundation for further study of Bordetella flagella and motility in the contexts of infection and transmission., (Copyright © 2019 Hoffman et al.)

Bordetella pertussis causes the disease whooping cough through coordinated control of virulence factors by the Bordetella virulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describe in vitro gene expression profiles of B. pertussis and other pathogens. In previous studies, we have analyzed the in vitro gene expression profiles of B. pertussis , and we hypothesize that the infection transcriptome profile in vivo is significantly different from that under laboratory growth conditions. To study the infection transcriptome of B. pertussis , we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-μm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing the in vitro and in vivo gene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical for B. pertussis survival in vivo Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile of B. pertussis during infection, and this method will facilitate efforts to understand how this pathogen causes infection. IMPORTANCE In vitro growth conditions for bacteria do not fully recapitulate the host environment. RNA sequencing transcriptome analysis allows for the characterization of the infection gene expression profiles of pathogens in complex environments. Isolation of the pathogen from infected tissues is critical because of the large amounts of host RNA present in crude lysates of infected organs. A filtration method was developed that enabled enrichment of the pathogen RNA for RNA-seq analysis. The resulting data describe the "infection transcriptome" of B. pertussis in the murine lung. This strategy can be utilized for pathogens in other hosts and, thus, expand our knowledge of what bacteria express during infection., (Copyright © 2019 Wong et al.)

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice., (Copyright © 2018 American Society for Microbiology.)

Pertussis (whooping cough), caused by Bordetella pertussis , is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species., (Copyright © 2017 American Society for Microbiology.)

Adenylate cyclase toxin (ACT) is an important Bordetella pertussis virulence factor that is not included in current acellular pertussis vaccines. We previously demonstrated that immunization with the repeat-in-toxin (RTX) domain of ACT elicits neutralizing antibodies in mice and discovered the first two antibodies to neutralize ACT activities by occluding the receptor-binding site. Here, we fully characterize these antibodies and their epitopes. Both antibodies bind ACT with low nanomolar affinity and cross-react with ACT homologues produced by B. parapertussis and B. bronchiseptica. Antibody M1H5 binds B. pertussis RTX 751 ∼100-fold tighter than RTX 751 from the other two species, while antibody M2B10 has similar affinity for all three variants. To initially map the antibody epitopes, we generated a series of ACT chimeras and truncation variants, which implicated the repeat blocks II-III. To identify individual epitope residues, we displayed randomly mutated RTX 751 libraries on yeast and isolated clones with decreased antibody binding by flow cytometry. Next-generation sequencing identified candidate epitope residues on the basis of enrichment of clones with mutations at specific positions. These epitopes form two adjacent surface patches on a predicted structural model of the RTX 751 domain, one for each antibody. Notably, the cellular receptor also binds within blocks II-III and shares at least one residue with the M1H5 epitope. The RTX 751 model supports the notion that the antibody and receptor epitopes overlap. These data provide insight into mechanisms of ACT neutralization and guidance for engineering more stable RTX variants that may be more appropriate vaccine antigens.

Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination., (Copyright © 2017 American Society for Microbiology.)

The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10-14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28-35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In addition to the ever-present concern of medical professionals about epidemics of infectious diseases, the relative ease of access and low cost of obtaining, producing, and disseminating pathogenic organisms or biological toxins mean that bioterrorism activity should also be considered when facing a disease outbreak. Utilization of whole-genome sequencing (WGS) in outbreak analysis facilitates the rapid and accurate identification of virulence factors of the pathogen and can be used to identify the path of disease transmission within a population and provide information on the probable source. Molecular tools such as WGS are being refined and advanced at a rapid pace to provide robust and higher-resolution methods for identifying, comparing, and classifying pathogenic organisms. If these methods of pathogen characterization are properly applied, they will enable an improved public health response whether a disease outbreak was initiated by natural events or by accidental or deliberate human activity. The current application of next-generation sequencing (NGS) technology to microbial WGS and microbial forensics is reviewed., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins' glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated., Results: Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB's rapid effects. gdTcdB did not clearly delay TcdA or TcdB's toxin-induced effects on macrophages., Conclusions: Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.

The adenylate cyclase toxin (ACT) of Bordetella pertussis intoxicates target cells by generating supraphysiologic levels of intracellular cyclic AMP (cAMP). Since ACT kills macrophages rapidly and potently, we asked whether ACT would also kill neutrophils. In fact, ACT prolongs the neutrophil life span by inhibiting constitutive apoptosis and preventing apoptosis induced by exposure to live B. pertussis. Imaging of B. pertussis-exposed neutrophils revealed that B. pertussis lacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-type B. pertussis does not, suggesting that ACT suppresses NET formation. Indeed, ACT inhibits formation of NETs by generating cAMP and consequently inhibiting the oxidative burst. Convalescent-phase serum from humans following clinical pertussis blocks the ACT-mediated suppression of NET formation. These studies provide novel insight into the phagocyte impotence caused by ACT, which not only impairs neutrophil function but also inhibits death of neutrophils by apoptosis and NETosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Pertussis is a worldwide public health threat. Bordetella pertussis produces multiple virulence factors that have been studied individually, and many have recently been found to have additional biological activities. Nevertheless, how they interact to cause the disease pertussis remains unknown. New animal models, particularly the infection of infant baboons with B. pertussis, are enabling longstanding questions about pertussis pathogenesis to be answered and new ones to be asked. Enhancing our understanding of pathogenesis will enable new approaches to the prevention and control of pertussis.

A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

Toxin A (TcdA) and toxin B (TcdB) of Clostridium difficile cause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g., Cxcl1 and Cxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses to C. difficile toxins.

Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ≈ 10(8) CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ≈ 10(8) CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ≈ 60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

Bordetella pertussis and Bordetella bronchiseptica establish respiratory infections with notorious efficiency. Our previous studies showed that the fhaB genes of B. pertussis and B. bronchiseptica, which encode filamentous hemagglutinin (FHA), are functionally interchangeable and provided evidence that FHA-deficient B. bronchiseptica induces more inflammation in the lungs of mice than wild-type B. bronchiseptica. We show here that the robust inflammatory response to FHA-deficient B. bronchiseptica is characterized by the early and sustained influx of interleukin-17 (IL-17)-positive neutrophils and macrophages and, at 72 h postinoculation, IL-17-positive CD4(+) T cells, suggesting that FHA allows the bacteria to suppress the development of an IL-17-mediated inflammatory response. We also show that the cyaA genes of B. pertussis and B. bronchiseptica, which encode adenylate cyclase toxin (ACT), are functionally interchangeable and that ACT, specifically its catalytic activity, is required for B. bronchiseptica to resist phagocytic clearance but is neither required for nor inhibitory of the induction of inflammation if bacteria are present in numbers sufficient to persist during the first 3 days postinoculation. Incubation of bone marrow-derived macrophages with a ΔcyaA strain caused decreased production of IL-1β and increased production of tumor necrosis factor alpha (TNF-α) and IL-12, while incubation with a ΔcyaA ΔfhaB strain caused increased production of IL-23. These data suggest that FHA and ACT both contribute to suppress the recruitment of neutrophils and the development of an IL-17-mediated immune response. To our knowledge, this is the first demonstration of a microbial pathogen suppressing IL-17-mediated inflammation in vivo as a strategy to evade innate immunity.

For many infectious diseases, novel treatment options are needed in order to address problems with cost, toxicity and resistance to current drugs. Systems biology tools can be used to gain valuable insight into pathogenic processes and aid in expediting drug discovery. In the past decade, constraint-based modeling of genome-scale metabolic networks has become widely used. Focusing on pathogen metabolic networks, we review in silico strategies used to identify effective drug targets and highlight recent successes as well as limitations associated with such computational analyses. We further discuss how accounting for the host environment and even targeting the host may offer new therapeutic options. These systems-level approaches are beginning to provide novel avenues for drug targeting against infectious agents., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.)

The adenylate cyclase toxin (ACT) of Bordetella pertussis does not require a receptor to generate intracellular cyclic AMP (cAMP) in a broad range of cell types. To intoxicate cells, ACT binds to the cell surface, translocates its catalytic domain across the cell membrane, and converts intracellular ATP to cAMP. In cells that express the integrin CD11b/CD18 (CR3), ACT is more potent than in CR3-negative cells. We find, however, that the maximum levels of cAMP accumulation inside CR3-positive and -negative cells are comparable. To better understand how CR3 affects the generation of cAMP, we used Chinese hamster ovary and K562 cells transfected to express CR3 and examined the steps in intoxication in the presence and absence of the integrin. The binding of ACT to cells is greater in CR3-expressing cells at all concentrations of ACT, and translocation of the catalytic domain is enhanced by CR3 expression, with ∼80% of ACT molecules translocating their catalytic domain in CR3-positive cells but only 25% in CR3-negative cells. Once in the cytosol, the unregulated catalytic domain converts ATP to cAMP, and at ACT concentrations >1,000 ng/ml, the intracellular ATP concentration is <5% of that in untreated cells, regardless of CR3 expression. This depletion of ATP prevents further production of cAMP, despite the CR3-mediated enhancement of binding and translocation. In addition to characterizing the effects of CR3 on the actions of ACT, these data show that ATP consumption is yet another concentration-dependent activity of ACT that must be considered when studying how ACT affects target cells.

Background: Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure., Results: We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis., Conclusions: This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.

Bacillus anthracis oedema toxin (ET) and Bordetella pertussis adenylate cyclase toxin (ACT) enter host cells and produce cAMP. To understand the cellular consequences, we exposed J774 cells to these toxins at ng ml(-1) (pM) concentrations, then followed cell number and changes in cell signalling pathways. Under these conditions, both toxins produce a concentration-dependent inhibition of cell proliferation without cytotoxicity. ET and ACT increase the proportion of cells in G(1) /G(0) and reduce S phase, such that a single addition of ET or ACT inhibits cell division for 3-6 days. Treatment with ET or ACT produces striking changes in proteins controlling cell cycle, including virtual elimination of phosphorylated ERK 1/2 and Cyclin D1 and increases in phospho-CREB and p27(Kip1) . Importantly, PD98059, a MEK inhibitor, elicits a comparable reduction in Cyclin D1 to that produced by the toxins and blocks proliferation. These data show that non-lethal concentrations of ET and ACT impose a prolonged block on the proliferation of J774 cells by impairment of the progression from G(1) /G(0) to S phase in a process involving cAMP-mediated increases in phospho-CREB and p27(Kip1) and reductions in phospho-ERK 1/2 and Cyclin D1. This phenomenon represents a new mechanism by which these toxins affect host cells., (© 2010 Blackwell Publishing Ltd.)

The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.

Bordetella pertussis causes a profound inflammatory response in lungs of infected individuals. The adenylate cyclase toxin (ACT) of B. pertussis is a potent enzyme that converts cytosolic ATP into cAMP, and is required for virulence in vivo. During infection, secreted ACT binds to macrophages utilizing the beta2 integrin, Mac-1 (CR3, CD11b/CD18), and subsequent intoxication by ACT inhibits essential antibacterial activities of macrophages. Additionally, Mac-1 has been reported to be a co-receptor for TLR4 required for the full induction of some LPS-responsive genes, including pro-inflammatory cyclooxygenase 2 (COX-2). We have examined the effect of ACT on COX-2 expression in HEK293T cells expressing Mac-1 and in murine macrophages. We report that ACT induces COX-2 in a manner that absolutely requires the catalytic activity of this enzyme and Mac-1 expression dramatically enhanced the sensitivity of cells to ACT-dependent COX-2 induction. The mechanism of COX-2 induction by ACT utilizes the cAMP-PKA-CREB-dependent pathway. Finally, ACT and TLR2 or TLR4 act synergistically to increase COX-2 expression. These data suggest that ACT contributes significantly to the inflammatory response induced by B. pertussis infection by augmenting COX-2 expression and provides evidence against the concept that ACT functions exclusively via its inhibitory effects on phagocytic leucocytes.

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in-answer-out results in < 30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 microl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

The signaling pathway by which luteinizing hormone (LH) acts on the somatic cells of vertebrate ovarian follicles to stimulate meiotic resumption in the oocyte requires a decrease in cAMP in the oocyte, but how cAMP is decreased is unknown. Activation of Gi family G proteins can lower cAMP by inhibiting adenylate cyclase or stimulating a cyclic nucleotide phosphodiesterase, but we show here that inhibition of this class of G proteins by injection of pertussis toxin into follicle-enclosed mouse oocytes does not prevent meiotic resumption in response to LH. Likewise, elevation of Ca2+ can lower cAMP through its action on Ca2+-sensitive adenylate cyclases or phosphodiesterases, but inhibition of a Ca2+ rise by injection of EGTA into follicle-enclosed mouse oocytes does not inhibit the LH response. Thus, neither of these well-known mechanisms of cAMP regulation can account for LH signaling to the oocyte in the mouse ovary.

The cytotoxic effect of adenylate cyclase (AC) toxin from Bordetella pertussis on host cells has been attributed to the production of supraphysiologic levels of cyclic AMP by the toxin. We have tested this hypothesis and show that at least two different mechanisms, cAMP accumulation/ATP depletion and oligomerization/pore formation, contribute, perhaps synergistically, to AC toxin-induced cytotoxicity. Wild-type (WT) AC toxin causes cell death associated with an increase in cAMP, a reduction in ATP, activation of caspases 3/7, and increased annexin V and TUNEL staining. In contrast, a non-acylated, enzymatically active, non-haemolytic form of AC toxin is able to increase cAMP, reduce ATP and elicit annexin V staining, but the decrease in ATP and the annexin staining are transient and there is minimal caspase activation, TUNEL staining and cell death. Mutant AC toxins defective in either enzymatic activity or the ability to deliver their enzymatic domain are able to kill J774 cells, without cAMP production, and with minimal caspase activation and TUNEL staining. Comparison of the potencies of WT toxin and those of mutants that only increase cAMP or only create transmembrane pores establishes that at least two mechanisms are contributory and that simply the production of cAMP is not enough to account for the cytotoxicity produced by AC toxin.

Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism.

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.

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.

Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the beta-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting betaTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca(2+) totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca(2+), which were blocked by nifedipine and the omission of extracellular Ca(2+). Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca(2+). PKA alpha, beta and gamma catalytic subunits (Calpha, Cbeta and Cgamma respectively) were identified in betaTC6 cells by confocal microscopy. Glucose and glucagon-like polypeptide-1 (GLP-1) caused translocation of Calpha to the nucleus and of Cbeta to the plasma membrane and the nucleus, but did not affect the distribution of Cgamma. In conclusion, glucose and GLP-1 amplify insulin secretion via cAMP production and PKAbeta activation.

Adenylate cyclase (AC) toxin from Bordetella pertussis is unusual in that, unlike most other members of the repeats-in-toxin family that are released into the extracellular milieu, it remains associated with the bacterial surface. In this study, we investigated the nature of the association of this toxin with the surface of B. pertussis. AC toxin was extracted from crude outer membrane preparations of B. pertussis with 8 M urea, but only partially with alkaline sodium carbonate and not at all with octylglucoside, suggesting that denaturation of the toxin is necessary for its removal from the membrane. B. pertussis mutants lacking filamentous haemagglutinin (FHA) released significantly more AC toxin into the medium, and AC toxin association with the bacterial surface was partially restored by expression of FHA from a plasmid, suggesting a role for FHA in surface retention of AC toxin. AC toxin distribution was unaffected by the absence of pertactin, or full-length lipopolysaccharide, or a defect in secretion of pertussis toxin. Using overlay and immunoprecipitation, we found that a direct physical association can occur between AC toxin and FHA. Combined, these findings suggest that FHA may play a role in AC toxin retention on the surface of B. pertussis and raise the possibility of an involvement of adherence mediated by FHA in delivery of AC toxin from the bacterium to the target cell.

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., (Copyright 2001 Academic Press.)

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.

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.

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.

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.

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.

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.

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.

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.

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.