The interaction of Bordetella pertussis adenylate cyclase toxin (CyaA) with complement receptor 3 (CR3, CD11b/CD18) involves N-linked oligosaccharide chains. To investigate the relative importance of the individual N-glycans of CR3 for toxin activity, the asparagine residues of the consensus N-glycosylation sites of CR3 were substituted with glutamine residues that cannot be glycosylated. Examination of CR3 mutant variants and mass spectrometry analysis of the N-glycosylation pattern of CR3 revealed that N-glycans located in the C-terminal part of the CD11b subunit are involved in binding and cytotoxic activity of CyaA. We suggest that these N-glycans form a defined clustered saccharide patch that enables multivalent contact of CR3 with CyaA, enhancing both affinity and specificity of the integrin-toxin interaction.

• MeSH
• adenylátcyklasový toxin genetika   metabolismus   MeSH
• antigeny CD11b chemie   metabolismus   MeSH
• antigeny CD18 chemie   metabolismus   MeSH
• asparagin genetika   MeSH
• Bordetella pertussis metabolismus   patogenita   MeSH
• glutamin genetika   MeSH
• glykosylace MeSH
• lidé MeSH
• makrofágový antigen 1 genetika   metabolismus   MeSH
• polysacharidy metabolismus   MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• Bordetella pertussis patogenita   MeSH
• buněčné linie MeSH
• interakce hostitele a patogenu * MeSH
• křečci praví MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, α(M)β(2) or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action.

• MeSH
• adenosintrifosfát chemie   MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• AMP cyklický biosyntéza   MeSH
• antigeny CD11b genetika   metabolismus   MeSH
• antigeny CD18 genetika   metabolismus   MeSH
• biologický transport fyziologie   MeSH
• Bordetella pertussis metabolismus   MeSH
• buněčné linie MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• fagocyty metabolismus   MeSH
• lidé MeSH
• makrofágový antigen 1 biosyntéza   genetika   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400-710 of CyaA as an "AC translocon" sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• antigen-1 spojený s lymfocytární funkcí metabolismus   MeSH
• Bordetella * MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cytosol metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I-V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132-1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562-1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295-1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.

• MeSH
• acylace MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• Bordetella pertussis patogenita   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• epitopy metabolismus   MeSH
• lidé MeSH
• makrofágový antigen 1 chemie   metabolismus   MeSH
• neutralizující protilátky metabolismus   MeSH
• proteinové domény MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• THP-1 buňky MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Komplement systém je systém kaskády mnohotvárných proteinů, který v sobě zahrnuje nespecifickou imunitní odpověď. Aktivace komplement systému nastává vlivem různých podnětů, jako je úraz, infekce, imunitní reakce. Jeho hlavní funkcí je aktivace obranných mechanismů, opsonizace (zvýšení účinnosti) proti cizorodým látkám, destrukce zbytků buněk. Zatím však zůstává kontroverzní souhrn různých složek komplementu, které se zabývají aktivací kaskády komplementu, a proto se může stát určitým nebezpečím vůči tkáním pro své cytotoxické vlastnosti. Různé studie v posledních letech ukázaly na příkladech závažných zánětlivých onemocněních, že při modulaci komplementu může vznikat určitý prospěšný ochranný účinek na orgány. Snaha o kontrolu komplement systému spočívá v podávání endogenních inhibitorů komplementu, tj. C1-inhibitoru (C1-INH) nebo v podávání rekombinovaných receptorů jako rozpustný komplement receptor 1 (rsCR1). Navíc protilátky proti proteinům (C3, C5), dále proti jejich aktivačním produktům (C5a) i proti receptoru komplementu 3 (CR, CR 18/11b) zprostředkovávají přilnavost leukocytů k cévnímu endotelu, reprezentují účinnou představu o modulaci komplementu. Vedle toho obsazení membrány lidskými komplement regulátory (DAF-CD 55, MCP-CD46 nebo CD59) v orgánech xenogenních dárců prokázalo účinnost vedoucí k prevenci odmítnutí xenogenních štěpů. Jak prokázaly pokusy na zvířatech, popsané postupy ochránily před vážným orgánovým poškozením při sepsi, při srdeční a střevní ischemii, při úrazu, ARDS, při zánětu ledvin a implantaci štěpů. S ohledem na současné klinické údaje by mohla inhibice komplementu představovat určitou terapeutickou strategii při kontrole směrování vážných stavů zánětu. Naše pokusy prokázaly ochranné účinky modulace komplementu s C1-INH a rs CR1 v modelu navozeného úrazu. S ohledem na možné snížení obrany hostitele je třeba posuzovat užití komplement inhibitorů velice opatrně.

The complement system is a multifactorial protein cascade system which is essentially involved in the early unspecific immune response. Activation of the complement system is achieved by a variety of stimuli e.g. trauma, infection or immunologic reactions. Its major function is the activation of cellular defense mechanisms, opsonisation of foreign particles and the destruction of target cells. While the impact of the different complement components for bacterial elimination still remains controversial, overwhelming activation of the complement cascade, however, can induce life threatening tissue damage due to the effective cytotoxic properties. In the last years a variety of studies demonstrated beneficial, organ protective effects of complement modulation in models of severe inflammation. Attempts to control the complement system include the application of endogenous complement inhibitors e.g. C1-Inhibitor (C1-INH) or the administration of recombinant complement receptors such as the soluble complement receptor 1 (rsCR1). Moreover antibodies against key proteins (C3, C5), against their activation products (C5a) or against complement receptor 3 (CR3, CD18/11b) mediated adhesion of leukocytes to the vascular endothelium, represent effective options of complement modulation. Besides this, insertion of membrane bound human complement regulators (DAF – CD55, MCP – CD46 or CD59) into xenogenic donor organs, has proven effectiveness to prevent xenograft rejection. The described interventions protected from severe organ damage in various animal models of sepsis, myocardial and intestinal ischaemia – reperfusion injury, ARDS, nephritis, and xenograft rejection. With respect to recent clinical data, complement inhibition could represent a useful therapeutic strategy to control overwhelming inflammation. Own experiments demonstrated protective effects of complement modulation with C1 INH and rsCR1 in a model of complement induced pulmonary injury. With respect to a potential impairment of host defense, however, the use of complement inhibitors must be considered carefully.

• MeSH
• aktivace komplementu MeSH
• imunita MeSH
• inaktivátory komplementu MeSH
• sepse MeSH

Bordetella adenylate cyclase toxin (CyaA) binds the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+) influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

• MeSH
• adenylátcyklasový toxin chemie   metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD18 metabolismus   MeSH
• Bordetella enzymologie   MeSH
• buněčná membrána enzymologie   mikrobiologie   MeSH
• cholesterol metabolismus   MeSH
• cytosol enzymologie   MeSH
• extracelulární prostor metabolismus   MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• makrofágy metabolismus   mikrobiologie   MeSH
• membránové mikrodomény enzymologie   mikrobiologie   MeSH
• myši MeSH
• talin metabolismus   MeSH
• terciární struktura proteinů MeSH
• U937 buňky MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

• MeSH
• akutní promyelocytární leukemie patologie   MeSH
• antigen-1 spojený s lymfocytární funkcí chemie   MeSH
• buněčná adheze účinky léků   fyziologie   MeSH
• cytochalasin B farmakologie   MeSH
• degranulace buněk * účinky léků   MeSH
• fibrinogen MeSH
• histamin farmakologie   MeSH
• indoly farmakologie   MeSH
• konformace proteinů účinky léků   MeSH
• kultivované buňky MeSH
• lidé MeSH
• makrofágový antigen 1 fyziologie   MeSH
• MAP kinasový signální systém účinky léků   MeSH
• messenger RNA biosyntéza   genetika   MeSH
• mitogenem aktivované proteinkinasy p38 fyziologie   MeSH
• N-formylmethionin-leucyl-fenylalanin farmakologie   MeSH
• nádorové buněčné linie MeSH
• neutrofily účinky léků   fyziologie   MeSH
• oximy farmakologie   MeSH
• piperaziny farmakologie   MeSH
• piperidiny farmakologie   MeSH
• pyridiny farmakologie   MeSH
• receptory histaminu fyziologie   MeSH
• receptory spřažené s G-proteiny agonisté   antagonisté a inhibitory   fyziologie   MeSH
• tvar buňky účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b(+)) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b(-) cells. The nonhemolytic AC(+) Hly(-) bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC(+) Hly(-) mutant also infected mouse lungs as efficiently as the parental AC(+) Hly(+) strain. Hence, elevation of cAMP in CD11b(-) cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10(7) CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• AMP cyklický metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• Bordetella pertussis patogenita   MeSH
• buněčná membrána metabolismus   MeSH
• dendritické buňky imunologie   MeSH
• fagocyty imunologie   MeSH
• hemolyziny metabolismus   MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pertuse mikrobiologie   MeSH
• plíce mikrobiologie   patologie   MeSH
• T-lymfocyty imunologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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