- MeSH
- biologie buňky * MeSH
- lidé MeSH
- mikrobiologie * MeSH
- sekreční systém typu VI metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- rozhovory MeSH
- Geografické názvy
- Česká republika MeSH
Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA
- MeSH
- adenylátcyklasový toxin farmakologie genetika metabolismus MeSH
- bakteriální proteiny farmakologie genetika metabolismus MeSH
- Bordetella enzymologie genetika MeSH
- erytrocytární membrána metabolismus MeSH
- financování organizované MeSH
- hemolýza genetika účinky léků MeSH
- missense mutace MeSH
- ovce MeSH
- signální transdukce genetika účinky léků MeSH
- substituce aminokyselin MeSH
- transport proteinů genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Bordetella adenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the alphaMbeta2 integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC- toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC- toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenylátcyklasový toxin toxicita MeSH
- AMP cyklický metabolismus MeSH
- antigeny CD11b metabolismus MeSH
- antigeny CD18 metabolismus MeSH
- Bordetella pertussis enzymologie imunologie MeSH
- buněčná smrt imunologie MeSH
- buněčné linie MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- cytotoxicita imunologická MeSH
- erytrocyty metabolismus MeSH
- financování organizované MeSH
- křečci praví MeSH
- monocyty enzymologie imunologie MeSH
- myši MeSH
- ovce MeSH
- permeabilita buněčné membrány imunologie MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- myši MeSH
- zvířata MeSH
