Abstract
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.
Highlights
The adenylate cyclase (CyaA) toxin is a major virulence factor produced by Bordetella pertussis, the causative agent of whooping cough, and is involved in the early stages of respiratory tract colonization.[1,2] CyaA, a 1706-residue long protein (Figure S1, Supporting Information), is a Repeat-in-ToXin (RTX)[3,4,5,6] multi-domain toxin.[7,8] Once secreted by B. pertussis, CyaA invades eukaryotic cells through an original molecular mechanism that involves a direct translocation of its N-terminal adenyl cyclase catalytic (AC) domain across the plasma membrane
We have previously shown that the P454 peptide, residues 454– 484 from the CyaA translocation region, exhibits membraneactive properties similar to antimicrobial peptides (AMPs).[58,59,60]
We show that substitutions of a few residues within the P454 segment are sufficient to fully abrogate www.advancedscience.com the delivery of the AC domain into the cell cytoplasm, without impairing toxin binding to target cells
Summary
The adenylate cyclase (CyaA) toxin is a major virulence factor produced by Bordetella pertussis, the causative agent of whooping cough, and is involved in the early stages of respiratory tract colonization.[1,2] CyaA, a 1706-residue long protein (Figure S1, Supporting Information), is a Repeat-in-ToXin (RTX)[3,4,5,6] multi-domain toxin.[7,8] Once secreted by B. pertussis, CyaA invades eukaryotic cells through an original molecular mechanism that involves a direct translocation of its N-terminal adenyl cyclase catalytic (AC) domain across the plasma membrane. S. Brier Biological NMR Technological Plateform Center for Technological Resources and Research Department of Structural Biology and Chemistry Institut Pasteur CNRS UMR3528 Paris 75015, France upon membrane interaction and induces a local destabilization of the lipid bilayer.[54,55,56,57] This property is likely essential for CyaA as deletion of the TR region (residues 384–489) encompassing the P454 segment, selectively abrogates the ability of the modified toxin to intoxicate target cells.[12]. We present structural models and crystal structures of the P454 peptide in complex with holo-CaM, and identified in P454, the amino acid residues that are critical for CaM-binding, membrane interaction, and destabilization Modifications of these residues within the full-length CyaA toxin are sufficient to fully and abrogate the translocation of the catalytic domain across the cell membrane. CaM is a key activator of the catalytic activity of CyaA inside cells, and acts as an essential cytosolic binder of the CyaA translocation region able to grab the polypeptide chain to favor its entry into target cells
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