Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin

Karst, Johanna C.; Barker, Robert; Devi, Usha; Swann, Marcus J.; Davi, Marilyne; Roser, Stephen J.; Ladant, Daniel; Chenal, Alexandre

doi:10.1074/jbc.m111.316166

Cited by 55 publications

(90 citation statements)

References 60 publications

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“…The CyaA monomer in solution is the functional state that partitions into the membrane mainly via hydrophobic forces, through its hydrophobic regions. Once CyaA is membrane-inserted, the translocation region may locally destabilize the lipid bilayer, favoring the passage of the catalytic domain across the plasma membrane (8,9). Meanwhile, CyaA monomers can interact and reorganize into oligomers within the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…ability to raise cAMP inside target cells) activities of the toxin were determined on sheep erythrocytes as described previously (8). Sheep erythrocytes were washed several times with buffer B and resuspended in this buffer at 5 ϫ 10 8 cells/ml.…”

Section: Methodsmentioning

confidence: 99%

“…Several domains can be identified in the C-terminal region. The so-called translocation region, spanning residues 400 -500, is crucial for the translocation of ACD across the plasma membrane (8) and exhibits properties related to membrane-active peptides (9). The hydrophobic region, spanning residues 500 -750, contains several hydrophobic segments predicted to adopt ␣-helical structures.…”

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confidence: 99%

“…It is believed that after binding of RD to the CD11b/CD18 receptor, the hydrophobic regions of CyaA may insert into the plasma membrane of the target cells. The catalytic domain is then delivered through the plasma membrane, possibly through a transient and local destabilization of the membrane integrity (8,9,42,43), to reach the cytosol, where, upon binding to the endogenous CaM, its enzymatic activity is stimulated to generate supraphysiologic levels of cAMP (37,44). Moreover, CyaA, after insertion into the membrane, can also form cation-selective pores, which impair membrane impermeability and ultimately cause cell lysis (6,40,45).…”

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confidence: 99%

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Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Karst

Enguéné

Cannella

et al. 2014

Journal of Biological Chemistry

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The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Karst

Enguéné

Cannella

et al. 2014

Journal of Biological Chemistry

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“…Delivery of the AC domain does not appear to proceed through the cation-selective pore formed by the hemolysin domain (25), but it requires structural integrity of the hydrophobic segments (24), and it seems that the RTX hemolysin domain (residues 374-1,706) harbors all structural information required for AC translocation (26). The region encompassing the helix 454-485 of ACT has been recently noted as possibly playing an important role in promoting translocation of the AC domain across the plasma membrane of target cells (27,28). A two-step model was proposed for AC translocation: in a first step, and upon ACT binding to its CD11b/ CD18 integrin receptor, a translocation-competent ACT conformer would conduct extracellular Ca 2+ ions across the plasma membrane, which would activate a calpain-mediated cleavage of talin, releasing the ACT-integrin complex from the cytoskeleton.…”

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confidence: 99%

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

González-Bullón

Uribe

Martı́n

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis. Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT-PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT-PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT-PLA activity thus emerges as novel target for therapeutic control of the disease.bacterial toxins | RTX toxin family | protein translocation | biological membranes | membrane remodeling

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A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

et al. 2021

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The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

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Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin

Cited by 55 publications

References 60 publications

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

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