Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4(+) and CD8(+) T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4(+)CD25(+)Foxp3(+) T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8(+) T cell proliferation and limited the induction of IFN-γ producing CD8(+) T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.
- MeSH
- adenylátcyklasový toxin farmakologie MeSH
- aktivace lymfocytů účinky léků MeSH
- Bordetella pertussis chemie MeSH
- buněčná adheze účinky léků MeSH
- buněčná smrt účinky léků MeSH
- CD antigeny metabolismus MeSH
- CD8-pozitivní T-lymfocyty cytologie účinky léků imunologie MeSH
- dendritické buňky cytologie účinky léků imunologie MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- pohyb buněk účinky léků MeSH
- proliferace buněk účinky léků MeSH
- regulační T-lymfocyty účinky léků MeSH
- rozpustnost MeSH
- toll-like receptory metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.
- MeSH
- adenylátcyklasový toxin farmakologie MeSH
- buněčné linie MeSH
- draslík metabolismus MeSH
- endocytóza účinky léků MeSH
- iontový transport účinky léků MeSH
- klathrin metabolismus MeSH
- makrofágy cytologie metabolismus MeSH
- membránové mikrodomény metabolismus MeSH
- myši MeSH
- permeabilita buněčné membrány účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- 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
Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated beta(2) integrin receptor CD11b/CD18 (alpha(M)beta(2), Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosidase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharide complex, whereas saccharides absent from integrin oligosaccharide chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the beta(2) integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli alpha-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharide chains of beta(2) integrin receptors. This sets a new paradigm for action of RTX cytotoxins.
- MeSH
- adenylátcyklasový toxin metabolismus MeSH
- antigeny CD11b metabolismus MeSH
- antigeny CD18 metabolismus MeSH
- bakteriální proteiny MeSH
- bakteriální toxiny metabolismus MeSH
- Bordetella enzymologie chemie patogenita MeSH
- financování organizované MeSH
- glykosylace MeSH
- hemolyziny MeSH
- lidé MeSH
- oligosacharidy metabolismus MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenylátcyklasový toxin genetika chemie izolace a purifikace metabolismus MeSH
- adenylátcyklasy metabolismus MeSH
- AMP cyklický metabolismus MeSH
- antigeny CD11b fyziologie MeSH
- biologický transport MeSH
- buněčná membrána fyziologie MeSH
- buněčné linie MeSH
- financování organizované MeSH
- hemolýza MeSH
- katalýza MeSH
- makrofágy fyziologie MeSH
- monocyty fyziologie MeSH
- mutageneze cílená MeSH
- myši MeSH
- ovce MeSH
- polymerázová řetězová reakce MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- substituce aminokyselin MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata 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
