The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC(-) toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.

• MeSH
• adenylátcyklasový toxin farmakokinetika   metabolismus   MeSH
• Bordetella enzymologie   MeSH
• endocytóza MeSH
• erytrocyty MeSH
• hemolýza účinky léků   MeSH
• missense mutace MeSH
• multimerizace proteinu MeSH
• ovce MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.

• MeSH
• adenylátcyklasový toxin imunologie   metabolismus   MeSH
• AMP cyklický imunologie   MeSH
• antigeny CD11b genetika   imunologie   MeSH
• antigeny CD18 genetika   imunologie   MeSH
• Bordetella pertussis enzymologie   imunologie   MeSH
• buněčná membrána imunologie   metabolismus   MeSH
• buněčné linie MeSH
• faktory depolymerizující aktin imunologie   metabolismus   MeSH
• financování organizované MeSH
• GTP-fosfohydrolasy imunologie   metabolismus   MeSH
• makrofágový antigen 1 imunologie   metabolismus   MeSH
• makrofágy imunologie   metabolismus   MeSH
• mikrofilamenta imunologie   metabolismus   MeSH
• myši MeSH
• neuropeptidy imunologie   metabolismus   MeSH
• pertuse enzymologie   imunologie   MeSH
• rac proteiny vázající GTP imunologie   metabolismus   MeSH
• rho proteiny vázající GTP imunologie   metabolismus   MeSH
• signální transdukce imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

• Publikační typ
• abstrakt z konference MeSH

• Publikační typ
• abstrakt z konference MeSH

Bordetella that infect mammals produce a multifunctional repeat in toxin (RTX) adenylate cyclase toxin known as CyaA, an excellent example of bacterial sophistication in subverting host defense. Recent reports show that interaction of CyaA with tracheal epithelial cells aids adhesion of Bordetella to ciliated mucosa and induces production of the pro-inflammatory cytokine interleukin, IL-6. Myeloid phagocytes, attracted to the site of infection are the target of freshly secreted CyaA that binds to the alpha(M)beta2 integrin (CD11b/CD18), penetrates cells and promptly suppresses their bactericidal functions by converting cellular ATP to cAMP. Such uncontrolled cAMP signaling can also drive CD11b-expressing immature dendritic cells into a semi-mature state, possibly hijacking them to shape the local adaptive immune response towards tolerance of the pathogen.

• MeSH
• adenylátcyklasový toxin metabolismus   toxicita   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD18 metabolismus   MeSH
• bakteriální adheze MeSH
• bakteriální proteiny metabolismus   toxicita   MeSH
• Bordetella imunologie   patogenita   MeSH
• dendritické buňky imunologie   MeSH
• epitelové buňky mikrobiologie   MeSH
• fagocyty imunologie   mikrobiologie   MeSH
• financování organizované MeSH
• infekce bakteriemi rodu Bordetella imunologie   mikrobiologie   MeSH
• infekce dýchací soustavy imunologie   mikrobiologie   MeSH
• interleukin-6 biosyntéza   MeSH
• lidé MeSH
• respirační sliznice mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH

Adenylate cyclase toxin (CyaA) of Bordetella pertussis penetrates the membrane of eukaryotic cells, producing high levels of intracellular cAMP, as well as hemolysis that results from the formation of cation-selective toxin channels in the membrane. Using several microscopical approaches we studied the effects of CyaA action on the morphology of sheep erythrocytes during early phases preceding lysis and examined localization of CyaA molecules within the erythrocyte membrane. CyaA induced a cascade of morphological changes of erythrocytes, such as shrinkage, formation of membrane projections, and blebs and swelling. The use of an enzymatically inactive CyaA-AC- toxoid that is unable to produce cAMP and of a CyaA-E581K mutant exhibiting higher hemolytic activity than with CyaA showed that the hemolytic activity is responsible for the induction of morphological changes of erythrocytes. Further, immunolabeling of inserted CyaA-232/FLAG molecules with specific anti-FLAG antibodies and IgG-gold particles indicated a clustered distribution of CyaA molecules in erythrocyte membrane. This was confirmed by immunofluorescence and confocal microscopy, which revealed uniform stoichiometry of CyaA clusters, suggesting CyaA binding into specific domains in erythrocyte membrane. Indeed, a decrease of CyaA binding after cholesterol depletion of erythrocytes suggests toxin targeting and binding to membrane microdomains (rafts). Copyright 2006 Wiley-Liss, Inc.

• MeSH
• adenylátcyklasový toxin analýza   toxicita   MeSH
• erytrocytární membrána chemie   ultrastruktura   MeSH
• erytrocyty chemie   účinky léků   ultrastruktura   MeSH
• financování organizované MeSH
• fluorescenční mikroskopie MeSH
• hemolýza MeSH
• imunoelektronová mikroskopie MeSH
• imunohistochemie MeSH
• konfokální mikroskopie MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• mikroskopie elektronová rastrovací transmisní MeSH
• mutace MeSH
• ovce MeSH
• substituce aminokyselin MeSH
• toxoidy metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH