A high-affinity calmodulin-binding site in the CyaA toxin translocation domain is essential for invasion into eukaryotic cells

Abstract

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. 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. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells.

The role of four cholesterol-recognition motifs localized between amino acid residues 400-550 in regulating translocation and lytic activity of Adenylate Cyclase Toxin

“…By contrast, the molecular mechanisms and the structural elements involved in AC domain translocation are less clear, and remain a matter of intense research. Several models have been postulated the last years [27,28]. One model posits that the ACT pore-forming activity is not implicated in the delivery of the AC domain across target cell membrane, and that on its way into the cytosol the translocating AC domain bypasses the cation-selective and lytic pore formed by ACT into the membrane [19,27].…”

“…Furthermore, structural integrity of all the transmembrane helices of the HD were shown to be essential for AC domain translocation across the plasma membrane of both CD11b+ and CD11bcells [19,23,24]. A second model posits that upon ACT insertion into the target cell membrane, a helical peptide extending from residue 454 to 484 interacts with the plasma membrane and destabilizes the lipid bilayer which would favour direct AC translocation across the lipid bilayer [28]. High affinity binding of that segment with calmodulin in the cell cytosol would then assist the irreversible translocation of the entire AC domain [28].…”

“…A second model posits that upon ACT insertion into the target cell membrane, a helical peptide extending from residue 454 to 484 interacts with the plasma membrane and destabilizes the lipid bilayer which would favour direct AC translocation across the lipid bilayer [28]. High affinity binding of that segment with calmodulin in the cell cytosol would then assist the irreversible translocation of the entire AC domain [28]. Data by others demonstrating the translocation of unrelated polypeptides fused to the ACT haemolysin moiety [29], weaken however reliability of this hypothesis.…”