Myeloid phagocytes have evolved to rapidly recognize invading pathogens and clear them through opsonophagocytic killing. The adenylate cyclase toxin (CyaA) of Bordetella pertussis and the edema toxin (ET) of Bacillus anthracis are both calmodulin-activated toxins with adenylyl cyclase activity that invade host cells and massively increase the cellular concentrations of a key second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP). However, the two toxins differ in the kinetics and mode of cell entry and generate different cAMP concentration gradients within the cell. While CyaA rapidly penetrates cells directly across their plasma membrane, the cellular entry of ET depends on receptor-mediated endocytosis and translocation of the enzymatic subunit across the endosomal membrane. We show that CyaA-generated membrane-proximal cAMP gradient strongly inhibits the activation and phosphorylation of Syk, Vav, and Pyk2, thus inhibiting opsonophagocytosis. By contrast, at similar overall cellular cAMP levels, the ET-generated perinuclear cAMP gradient poorly inhibits the activation and phosphorylation of these signaling proteins. Hence, differences in spatiotemporal distribution of cAMP produced by the two adenylyl cyclase toxins differentially affect the opsonophagocytic signaling in myeloid phagocytes.

• Klíčová slova
• 3′,5′-cyclic adenosine monophosphate (cAMP), Pyk2, Syk, Vav, adenylate cyclase toxin, edema toxin, opsonophagocytosis, phagocytes, signaling pathway,
• MeSH
• adenylátcyklasový toxin toxicita   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny bakteriální toxicita   MeSH
• bakteriální toxiny toxicita   MeSH
• časoprostorová analýza MeSH
• fagocytóza účinky léků   MeSH
• fagocyty účinky léků   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• mikrofilamenta účinky léků   MeSH
• opsoniny farmakologie   MeSH
• receptory imunologické metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• THP-1 buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• AMP cyklický MeSH
• anthrax toxin MeSH Prohlížeč
• antigeny bakteriální MeSH
• bakteriální toxiny MeSH
• opsonin receptor MeSH Prohlížeč
• opsoniny MeSH
• receptory imunologické MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of pathogenic Bordetellae delivers its adenylyl cyclase (AC) enzyme domain into the cytosol of host cells and catalyzes uncontrolled conversion of cellular ATP to cAMP. In parallel, the toxin forms small cation-selective pores that permeabilize target cell membrane and account for the hemolytic activity of CyaA on erythrocytes. The pore-forming domain of CyaA is predicted to consist of five transmembrane α-helices, of which the helices I, III, IV and V have previously been characterized. We examined here the α-helix II that is predicted to form between residues 529 to 549. Substitution of the glycine 531 residue by a proline selectively reduced the hemolytic capacity but did not affect the AC translocating activity of the CyaA-G531P toxin. In contrast, CyaA toxins with alanine 538 or 546 replaced by diverse residues were selectively impaired in the capacity to translocate the AC domain across cell membrane but remained fully hemolytic. Such toxins, however, formed pores in planar asolectin bilayer membranes with a very low frequency and with at least two different conducting states. The helix-breaking substitution of alanine 538 by a proline residue abolished the voltage-activated increase of membrane activity of CyaA in asolectin bilayers. These results reveal that the predicted α-helix comprising the residues 529 to 549 plays a key role in CyaA penetration into the target plasma membrane and pore-forming activity of the toxin.

• MeSH
• adenylátcyklasový toxin chemie   genetika   toxicita   MeSH
• Bordetella enzymologie   MeSH
• buněčná membrána účinky léků   MeSH
• erytrocyty účinky léků   MeSH
• hemolýza MeSH
• konformace proteinů, alfa-helix MeSH
• kultivované buňky MeSH
• myši MeSH
• ovce MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH