Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis

Two juxtaposed CRAC-CARC motifs at the N-terminus of the predicted transmembrane-helix II essential for the lytic activity and translocation of Bordetella

Trabajo presentado al European Workshop on Bacterial Protein Toxins (ETOX 21), celebrado en Alemania en junio de 2021

Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes

Adenylate Cyclase Toxin (ACT or CyaA) is one of the important virulence factors secreted by Bordetella pertussis, the bacterium causative of whooping cough. ACT debilitates host defenses by production of unregulated levels of cAMP into the cell cytosol upon delivery of its N-terminal domain with adenylate cyclase activity (AC domain) and by forming pores in the plasma membrane of macrophages. Binding of soluble toxin monomers to the plasma membrane of target cells and conversion into membrane-integrated proteins are the first and last step for these toxin activities; however, the molecular determinants in the protein or the target membrane that govern this conversion to an active toxin form are fully unknown. It was previously reported that cytotoxic and cytolytic activities of ACT depend on membrane cholesterol. Here we show that ACT specifically interacts with membrane cholesterol, and find in two membrane-interacting ACT domains, four cholesterol-binding motifs that are essential for AC domain translocation and lytic activities. We hypothesize that direct ACT interaction with membrane cholesterol through those four cholesterol-binding motifs drives insertion and stabilizes the transmembrane topology of several helical elements that ultimately build the ACT structure for AC delivery and pore-formation, thereby explaining the cholesterol-dependence of the ACT activities. The requirement for lipid-mediated stabilization of transmembrane helices appears to be a unifying mechanism to modulate toxicity in pore-forming toxins.This research was funded by the Spanish Ministerio de Economía y Competitividad [grant number BFU2017–82758-P] and by the Basque Government [grant number IT1264-19]. J.A. was recipient of a fellowship from the University of Basque Country (UPV/EHU). RA holds a contract funded by the Fundación Biofisika Bizkaia.Peer reviewe

Characterization of the Intrinsic Phospholipase A1 Activity of <i>Bordetella pertussis</i> 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&#8217;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

Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins

Permeabilization of the plasma membrane represents an important threat for any cell, since it compromises its viability by disrupting cell homeostasis. Numerous pathogenic bacteria produce pore-forming toxins that break plasma membrane integrity and cause cell death by colloid-osmotic lysis. Eukaryotic cells, in turn, have developed different ways to cope with the effects of such membrane piercing. Here, we provide a short overview of the general mechanisms currently proposed for plasma membrane repair, focusing more specifically on the cellular responses to membrane permeabilization by pore-forming toxins and presenting new data on the effects and cellular responses to the permeabilization by an RTX (repeats in toxin) toxin, the adenylate cyclase toxin-hemolysin secreted by the whooping cough bacterium Bordetella pertussis, which we have studied in the laboratory

Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology

Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered

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 Bordetella pertussis 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.This study was supported by grants from the Basque Government (Grupos Consolidados IT849‐13 and IT1264‐19) and grants from the Spanish Ministerio de Economía y Competitividad (BFU2014‐53469P to MTA A, and BFU‐2017‐82758‐P (AEI/FEDER, UE) to HO, AE was recipient of a fellowship from the University of the Basque Country (UPV/EH). DGB was recipient of a fellowship from the Bizkaia Biophysics Foundation

Advantages and versatility of fluorescence-based methodology to characterize the functionality of LDLR and class mutation assignment.

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with 125I-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the 125I methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than 125I-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations