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.

• Klíčová slova
• AC domain translocation, AC translocon, Bordetella pertussis, CyaA, Escherichia coli (E. coli), HlyA, RTX toxin, acylation, acyltransferase, bacterial toxin, complement receptor 3 (CR3,), fatty acid, fatty acyl, integrin, protein acylation, protein translocation,
• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• antigen-1 spojený s lymfocytární funkcí metabolismus   MeSH
• Bordetella * MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cytosol metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• antigen-1 spojený s lymfocytární funkcí MeSH
• makrofágový antigen 1 MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) plays a crucial role in virulence and airway colonization capacity of the whooping cough agent Bordetella pertussis. The toxin penetrates target cell membranes and exhibits three distinct biological activities. A population of CyaA conformers forms small cation-selective pores that permeabilize the cell membrane for potassium efflux, which can provoke colloid-osmotic (oncotic) cell lysis. The other two activities are due to CyaA conformers that transiently form calcium influx conduits in the target cell membrane and translocate the adenylate cyclase (AC) enzyme into cytosol of cells. A fourth putative biological activity has recently been reported; an intrinsic phospholipase A (PLA) activity was claimed to be associated with the CyaA polypeptide and be involved in the mechanism of translocation of the AC enzyme polypeptide across cell membrane lipid bilayer. However, the conclusions drawn by the authors contradicted their own results and we show them to be erroneous. We demonstrate that highly purified CyaA is devoid of any detectable phospholipase A1 activity and that contrary to the published claims, the two putative conserved phospholipase A catalytic residues, namely the Ser606 and Asp1079 residues, are not involved in the process of membrane translocation of the AC domain of CyaA across target membranes.

• Klíčová slova
• AC domain translocation, adenylate cyclase toxin, phospholipase A activity,
• MeSH
• adenylátcyklasový toxin metabolismus   toxicita   MeSH
• Bordetella pertussis MeSH
• buněčné linie MeSH
• erytrocyty MeSH
• fosfolipasy A metabolismus   MeSH
• hemolýza MeSH
• kyselina asparagová MeSH
• myši MeSH
• ovce MeSH
• serin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• fosfolipasy A MeSH
• kyselina asparagová MeSH
• serin MeSH