Adenylate Cyclase Toxin Research Articles

Retargeting from the CR3 to the LFA-1 receptor uncovers the adenylyl cyclase enzyme–translocating segment of Bordetella adenylate cyclase toxin

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400-710 of CyaA as an "AC translocon" sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.

Development of a targeted nanoLC-MS/MS method for quantitation of residual toxins from Bordetella pertussis

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis) infection. Pertussis pathogenesis is driven by cell-surface adhesion proteins and secreted toxins; some of which have been harnessed for their immunogenic properties as purified antigen components in acellular vaccines. Two of these virulence factors, adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), are protein toxins with potential for co-purification, and therefore must be monitored as process-related impurities during the development of acellular Pertussis vaccine candidates. Here we describe the development of a targeted nanoLC-MS/MS method for absolute quantitation of ACT and DNT in process intermediates from acellular Pertussis antigen purification. Starting from an in silico digest of the toxin sequences, a synthetic peptide screening approach was applied to systematically evaluate candidate sequences as surrogates for protein quantitation. Following refinement to a subset of sequences, absolutely quantified heavy-labelled (AQUA) peptides were implemented in a parallel reaction monitoring (PRM) workflow with limits of detection (LOD) and quantitation (LOQ) in the 12.5–25 amol (2−4 ng/mL) range on-column. In this work, we highlight a ‘standards-driven’ approach to surrogate peptide selection for protein quantitation. This strategy can be broadly applied in the absence of purified reference material and accelerate quantitative LC–MS method development across multiple sample matrices.

Functional and structural consequences of epithelial cell invasion by Bordetella pertussis adenylate cyclase toxin.

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

Negative charge of the AC-to-Hly linking segment modulates calcium-dependent membrane activities of Bordetella adenylate cyclase toxin

Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting ‘AC-to-Hly-linking segment’ (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411–490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411–490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA411–490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the ‘AC-to-Hly-linking segment’ (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.

Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor.

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.IMPORTANCEBordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

Membrane Activity and Channel Formation of the Adenylate Cyclase Toxin (CyaA) of Bordetella pertussis in Lipid Bilayer Membranes.

The Gram-negative bacterium Bordetella pertussis is the cause of whooping cough. One of its pathogenicity factors is the adenylate cyclase toxin (CyaA) secreted by a Type I export system. The 1706 amino acid long CyaA (177 kDa) belongs to the continuously increasing family of repeat in toxin (RTX) toxins because it contains in its C-terminal half a high number of nine-residue tandem repeats. The protein exhibits cytotoxic and hemolytic activities that target primarily myeloid phagocytic cells expressing the αMβ2 integrin receptor (CD11b/CD18). CyaA represents an exception among RTX cytolysins because the first 400 amino acids from its N-terminal end possess a calmodulin-activated adenylate cyclase (AC) activity. The entry of the AC into target cells is not dependent on the receptor-mediated endocytosis pathway and penetrates directly across the cytoplasmic membrane of a variety of epithelial and immune effector cells. The hemolytic activity of CyaA is rather low, which may have to do with its rather low induced permeability change of target cells and its low conductance in lipid bilayer membranes. CyaA forms highly cation-selective channels in lipid bilayers that show a strong dependence on aqueous pH. The pore-forming activity of CyaA but not its single channel conductance is highly dependent on Ca2+ concentration with a half saturation constant of about 2 to 4 mM.

Single Molecule Force Spectroscopy Reveals the Mechanical Design Governing the Efficient Translocation of the Bacterial Toxin Protein RTX.

The efficient translocation of the bacterial toxin adenylate cyclase toxin (CyaA) from the bacterial cytosol to the extracellular environment by the type 1 secretion system (T1SS) is essential for the toxin to function. To understand the molecular features that are responsible for the efficient translocation of CyaA, here we used optical tweezers to investigate the mechanical properties and conformational dynamics of the RTX domain of CyaA at the single molecule level. Our results revealed that apo-RTX behaves like an ideal random coil. This property allows the T1SS to translocate RTX without overcoming the enthalpic resistance. In contrast, the folded holo-RTX is mechancially stable, and its folding occurs in a vectorial, cotranslocational fashion starting from its C-terminus. Moreover, our results showed that the folding of holo-RTX generates a stretching force, which can further facilitate the translocation of RTX. Our results highlight the important role played by the Ca2+-triggered folding of RTX in the translocation of RTX and provide mechanistic insights into the mechanical design that governs the efficient translocation of RTX.

Irreversible versus repairable membrane poration: differences in permeabilization elicited by Bordetella Adenylate Cyclase Toxin and its hemolysin domain in macrophages.

Rapid plasma membrane repair in response to pore-forming toxins is crucial for cell survival, but the molecular mechanisms employed by eukaryotic nucleated cells to maintain membrane integrity and the specificities of such pathways remain poorly understood. Here, we have explored the permeabilization elicited by the Bordetellapertussis adenylate cyclase toxin, a 200-kDa protein toxin with α-helical pore-forming domain that forms pores of tunable size, and evaluated the response of target macrophages to such toxin poration. We show here that the response and the fate of target macrophages depend on toxin pore width. We find that the toxin's hemolysin moiety induces a transient membrane permeabilization by forming wide enough pores allowing Ca2+ influx into the target cell cytosol. This activates a Ca2+ -dependent cellular response involving exocytosis and endocytosis steps eliminating toxin pores and restoring membrane integrity. In contrast, the full-length native toxin, at low concentrations, forms very small pores that cause insidious perturbation of cell ion homeostasis that escapes control by the macrophage membrane repair response, eventually leading to cell death. Our data reveal that permeability to Ca2+ and ATP are key elements in the membrane repair pathway for eliminating α-helical pores of bacterial origin.

Channel Formation by LktA of Mannheimia (Pasteurella) haemolytica in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins.

Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system.

Bordetella Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells.

Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing B. pertussis bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression on in vitro differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated in vitro The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens.IMPORTANCE Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agent Bordetella pertussis The adenylate cyclase toxin (CyaA) mediates immune evasion of B. pertussis by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.

Post-translational acylation controls the folding and functions of the CyaA RTX toxin.

The adenylate cyclase (CyaA) toxin is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough. CyaA is synthetized as a pro-toxin, pro-CyaA, and converted into its cytotoxic form upon acylation of two lysines. After secretion, CyaA invades eukaryotic cells and produces cAMP, leading to host defense subversion. To gain further insights into the effect of acylation, we compared the functional and structural properties of pro-CyaA and CyaA proteins. HDX-MS results show that the refolding process of both proteins upon progressive urea removal is initiated by calcium binding to the C-terminal RTX domain. We further identified a critical hydrophobic segment, distal from the acylation region, that folds at higher urea concentration in CyaA than in pro-CyaA. Once refolded into monomers, CyaA is more compact and stable than pro-CyaA, due to a complex set of interactions between domains. Our HDX-MS data provide direct evidence that the presence of acyl chains in CyaA induces a significant stabilization of the apolar segments of the hydrophobic domain and of most of the acylation region. We propose a refolding model dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Therefore, CyaA acylation is not only critical for cell intoxication, but also for protein refolding into its active conformation. Our data shed light on the complex relationship between post-translational modifications, structural disorder and protein folding. Coupling calcium-binding and acylation-driven folding is likely pertinent for other repeat-in-toxin cytolysins produced by many Gram-negative bacterial pathogens.-O'Brien, D. P., Cannella, S. E., Voegele, A., Raoux-Barbot, D., Davi, M., Douché, T., Matondo, M., Brier, S., Ladant, D., Chenal, A. Post-translational acylation controls the folding and functions of the CyaA RTX toxin.

Distinct Spatiotemporal Distribution of Bacterial Toxin-Produced Cellular cAMP Differentially Inhibits Opsonophagocytic Signaling.

Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3’,5’-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.

Membrane Permeabilization by Pore-Forming RTX Toxins: What Kind of Lesions Do These Toxins Form?

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. The pore-forming cytolysins of the RTX (repeats in toxin) family belong to a steadily increasing family of proteins characterized by having in their primary sequences a number of glycine- and aspartate-rich nonapeptide repeats. They are secreted by a variety of Gram-negative bacteria and form ion-permeable pores in several cell types, such as immune cells, epithelial cells, or erythrocytes. Pore-formation by RTX-toxins leads to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. The pores formed in lipid bilayers by the RTX-toxins share some common properties such as cation selectivity and voltage-dependence. Hemolytic and cytolytic RTX-toxins are important virulence factors in the pathogenesis of the producing bacteria. And hence, understanding the function of these proteins at the molecular level is critical to elucidating their role in disease processes. In this review we summarize the current state of knowledge on pore-formation by RTX toxins, and include recent results from our own laboratory regarding the pore-forming activity of adenylate cyclase toxin (ACT or CyaA), a large protein toxin secreted by Bordetella pertussis, the bacterium causative of whooping cough.

Membrane Permeabilization by Bordetella Adenylate Cyclase Toxin Involves Pores of Tunable Size.

RTX (Repeats in ToXin) pore-forming toxins constitute an expanding family of exoproteins secreted by many Gram-negative bacteria and involved in infectious diseases caused by said pathogens. Despite the relevance in the host/pathogen interactions, the structure and characteristics of the lesions formed by these toxins remain enigmatic. Here, we capture the first direct nanoscale pictures of lytic pores formed by an RTX toxin, the Adenylate cyclase (ACT), secreted by the whooping cough bacterium Bordetella pertussis. We reveal that ACT associates into growing-size oligomers of variable stoichiometry and heterogeneous architecture (lines, arcs, and rings) that pierce the membrane, and that, depending on the incubation time and the toxin concentration, evolve into large enough “holes” so as to allow the flux of large molecular mass solutes, while vesicle integrity is preserved. We also resolve ACT assemblies of similar variable stoichiometry in the cell membrane of permeabilized target macrophages, proving that our model system recapitulates the process of ACT permeabilization in natural membranes. Based on our data we propose a non-concerted monomer insertion and sequential mechanism of toroidal pore formation by ACT. A size-tunable pore adds a new regulatory element to ACT-mediated cytotoxicity, with different pore sizes being putatively involved in different physiological scenarios or cell types.

Residues 529 to 549 participate in membrane penetration and pore-forming activity of the Bordetella adenylate cyclase toxin

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.

The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro.

The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity.

Characterization of the Intrinsic Phospholipase A1 Activity of Bordetella pertussis Adenylate Cyclase Toxin.

Adenylate cyclase toxin (ACT, CyaA) is one of the important virulence factors secreted by the whooping cough bacterium Bordetella pertussis, and it is essential for the colonization of the human respiratory tract by this bacterium. Cytotoxicity by ACT results from the synergy between toxin’s two main activities, production of supraphysiological cAMP levels by its N-terminal adenylate cyclase domain (AC domain), and cell membrane permeabilization, induced by its C-terminal pore-forming domain (hemolysin domain), which debilitate the host defenses. In a previous study we discovered that purified ACT is endowed with intrinsic phospholipase A1 (PLA) activity and that Ser in position 606 of the ACT polypeptide is a catalytic site for such hydrolytic activity, as part of G-X-S-X-G catalytic motif. Recently these findings and our conclusions have been directly questioned by other authors who claim that ACT-PLA activity does not exist. Here we provide new data on ACT phospholipase A1 characteristics. Based on our results we reaffirm our previous conclusions that ACT is endowed with PLA activity; that our purified ACT preparations are devoid of any impurity with phospholipase A activity; that ACT-S606A is a PLA-inactive mutant and thus, that Ser606 is a catalytic site for the toxin hydrolytic activity on phospholipids, and that ACT-PLA activity is involved in AC translocation.

Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens

Despite wide spread vaccination, the public health burden of pertussis remains substantial. Current acellular pertussis vaccines comprise upto five Bordetella pertussis (Bp) antigens. Performing an ELISA to quantify antibody for each antigen is laborious and challenging to apply to pediatric samples where serum volume may be limited. We developed a microsphere based multiplex antibody capture assay (MMACA) to quantify antibodies to five pertussis antigens; pertussis toxin, pertactin, filamentous hemagglutinin and fimbrial antigens 2/3, and adenylate cyclase toxin in a single reaction (5-plex) with a calibrated reference standard, QC reagents and SAS® based data analysis program. The goodness of fit (R2) of the standard curves for five analytes was ≥0.99, LLOQ 0.04–0.15 IU or AU/mL, accuracy 1.9%–23.8% (%E), dilutional linearity slopes 0.93–1.02 and regression coefficients r2 = 0.91–0.99. MMACA had acceptable precision within a median CV of 16.0%-22.8%. Critical reagents, antigen conjugated microsphere and reporter antibody exhibited acceptable (<12.3%) lot-lot variation. MMACA can be completed in <3 h, requires low serum volume (5μL/multiplex assay) and has fast data turnaround time (<1 min). MMACA has been successfully developed and validated as a sensitive, specific, robust and rugged method suitable for simultaneous quantification of anti-Bp antibodies in serum, plasma and DBS.

Differential regulation of actin-activated nucleotidyl cyclase virulence factors by filamentous and globular actin

Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.

Translocation and calmodulin-activation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis.

The adenylate cyclase toxin (CyaA) is a multi-domain protein secreted by Bordetella pertussis, the causative agent of whooping cough. CyaA is involved in the early stages of respiratory tract colonization by Bordetella pertussis. CyaA is produced and acylated in the bacteria, and secreted via a dedicated secretion system. The cell intoxication process involves a unique mechanism of transport of the CyaA toxin catalytic domain (ACD) across the plasma membrane of eukaryotic cells. Once translocated, ACD binds to and is activated by calmodulin and produces high amounts of cAMP, subverting the physiology of eukaryotic cells. Here, we review our work on the identification and characterization of a critical region of CyaA, the translocation region, required to deliver ACD into the cytosol of target cells. The translocation region contains a segment that exhibits membrane-active properties, i.e. is able to fold upon membrane interaction and permeabilize lipid bilayers. We proposed that this region is required to locally destabilize the membrane, decreasing the energy required for ACD translocation. To further study the translocation process, we developed a tethered bilayer lipid membrane (tBLM) design that recapitulate the ACD transport across a membrane separating two hermetic compartments. We showed that ACD translocation is critically dependent on calcium, membrane potential, CyaA acylation and on the presence of calmodulin in the trans compartment. Finally, we describe how calmodulin-binding triggers key conformational changes in ACD, leading to its activation and production of supraphysiological concentrations of cAMP.