title: Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin.
abstract: The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Jiri
authors: J
authors: Masin
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.|Faculty of Science, Charles University, Prague, Czech Republic.
authors: Adriana
authors: A
authors: Osickova
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Anna
authors: A
authors: Sukova
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.|Faculty of Science, Charles University, Prague, Czech Republic.
authors: Radovan
authors: R
authors: Fiser
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Petr
authors: P
authors: Halada
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Ladislav
authors: L
authors: Bumba
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Irena
authors: I
authors: Linhartova
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Radim
authors: R
authors: Osicka
authors: Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic.
authors: Peter
authors: P
authors: Sebo
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics; D001886:Bordetella pertussis / Q000737:chemistry / Q000472:pathogenicity; D002463:Cell Membrane Permeability / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D054353:Perforin / Q000737:chemistry; D014917:Whooping Cough / Q000235:genetics* / Q000382:microbiology / Q000473:pathology
chemical_list: D037361:Adenylate Cyclase Toxin; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D054353:Perforin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases