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
- 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
