• MeSH
• adenylátcyklasový toxin MeSH
• antigeny CD11b imunologie   MeSH
• antigeny CD18 imunologie   MeSH
• Bordetella pertussis patogenita   MeSH
• faktory virulence rodu Bordetella MeSH
• integriny imunologie   MeSH
• pertusový toxin MeSH

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) β, a GPI-anchored protein belonging to the folate receptor family. As FRβ shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRβ, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRβ in the plasma membrane of human FRβ(+) macrophages and FRβ-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRβ: that is, we report functional interactions of FRβ with receptors mediating cellular adhesion, in particular the CD11b/CD18 β2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRβ(+) human primary macrophages and THP-1 cells to collagen in comparison with their FRβ(-) counterparts. We further show that FRβ is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRβ as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen.

• MeSH
• antigeny CD11b fyziologie   MeSH
• antigeny CD18 fyziologie   MeSH
• buněčná adheze MeSH
• folátový receptor 2 fyziologie   MeSH
• kolagen farmakologie   MeSH
• kultivované buňky MeSH
• kyselina listová metabolismus   MeSH
• lidé MeSH
• makrofágy fyziologie   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The pertussis agent Bordetella pertussis produces a number of virulence factors, of which the filamentous hemagglutinin (FhaB) plays a role in B. pertussis adhesion to epithelial and phagocytic cells. Moreover, FhaB was recently found to play a crucial role in nasal cavity infection and B. pertussis transmission to new hosts. The 367 kDa FhaB protein translocates through an FhaC pore to the outer bacterial surface and is eventually processed to a ~220 kDa N-terminal FHA fragment by the SphB1 protease. A fraction of the mature FHA then remains associated with bacterial cell surface, while most of FHA is shed into the bacterial environment. Previously reported indirect evidence suggested that FHA, or its precursor FhaB, may bind the β2 integrin CD11b/CD18 of human macrophages. Therefore, we assessed FHA binding to various cells producing or lacking the integrin and show that purified mature FHA does not bind CD11b/CD18. Further results then revealed that the adhesion of B. pertussis to cells does not involve an interaction between the bacterial surface-associated FhaB and/or mature FHA and the β2 integrin CD11b/CD18. In contrast, FHA binding was strongly inhibited at micromolar concentrations of heparin, corroborating that the cell binding of FHA is ruled by the interaction of its heparin-binding domain with sulfated glycosaminoglycans on the cell surface.

• MeSH
• antigeny CD18 MeSH
• bakteriální adheze MeSH
• bakteriální adheziny metabolismus   MeSH
• Bordetella pertussis * metabolismus   MeSH
• faktory virulence rodu Bordetella MeSH
• glykosaminoglykany MeSH
• hemaglutininy metabolismus   MeSH
• heparin MeSH
• integriny MeSH
• lidé MeSH
• makrofágový antigen 1 MeSH
• pertuse * MeSH
• proteasy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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 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

The aim of this study was to compare two methods for quantification of changes in intracellular potassium concentration (decrease from ∼140 to ∼20mM) due to the action of a pore-forming toxin, the adenylate cyclase toxin (CyaA) from the pathogenic bacterium Bordetella pertussis. CyaA was incubated with stably transfected K1 Chinese hamster ovary cells expressing the toxin receptor CD11b/CD18 and the decrease in potassium concentration in the cells was followed by inductively coupled plasma mass spectrometry (ICP-MS). It is shown that this method is superior in terms of sensitivity, accuracy, and temporal resolution over the method employing the potassium-binding benzofuran isophthalate-acetoxymethyl ester fluorescent indicator. The ICP-MS procedure was found to be a reliable and straightforward analytical approach enabling kinetic studies of CyaA action at physiologically relevant toxin concentrations (<1000ng/ml) in biological microsamples.

• MeSH
• adenylátcyklasový toxin toxicita   MeSH
• antigeny CD11b genetika   MeSH
• antigeny CD18 genetika   MeSH
• Bordetella pertussis enzymologie   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• draslík chemie   metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• intracelulární prostor účinky léků   metabolismus   MeSH
• křečci praví MeSH
• lidé MeSH
• transfekce 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

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

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

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