Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity.
We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin. (+info)
Distinct roles for Galphai2, Galphai3, and Gbeta gamma in modulation offorskolin- or Gs-mediated cAMP accumulation and calcium mobilization by dopamine D2S receptors.
Previous studies have shown that a single G protein-coupled receptor can regulate different effector systems by signaling through multiple subtypes of heterotrimeric G proteins. In LD2S fibroblast cells, the dopamine D2S receptor couples to pertussis toxin (PTX)-sensitive Gi/Go proteins to inhibit forskolin- or prostaglandin E1-stimulated cAMP production and to stimulate calcium mobilization. To analyze the role of distinct Galphai/o protein subtypes, LD2S cells were stably transfected with a series of PTX-insensitive Galphai/o protein Cys --> Ser point mutants and assayed for D2S receptor signaling after PTX treatment. The level of expression of the transfected Galpha mutant subunits was similar to the endogenous level of the most abundant Galphai/o proteins (Galphao, Galphai3). D2S receptor-mediated inhibition of forskolin-stimulated cAMP production was retained only in clones expressing mutant Galphai2. In contrast, the D2S receptor utilized Galphai3 to inhibit PGE1-induced (Gs-coupled) enhancement of cAMP production. Following stable or transient transfection, no single or pair set of mutant Galphai/o subtypes rescued the D2S-mediated calcium response following PTX pretreatment. On the other hand, in LD2S cells stably transfected with GRK-CT, a receptor kinase fragment that specifically antagonizes Gbeta gamma subunit activity, D2S receptor-mediated calcium mobilization was blocked. The observed specificity of Galphai2 and Galphai3 for different states of adenylyl cyclase activation suggests a higher level of specificity for interaction of Galphai subunits with forskolin- versus Gs-activated states of adenylyl cyclase than has been previously appreciated. (+info)
GABA(B) receptor-mediated stimulation of adenylyl cyclase activity in membranes of rat olfactory bulb.
Previous studies have shown that GABA(B) receptors facilitate cyclic AMP formation in brain slices likely through an indirect mechanism involving intracellular second messengers. In the present study, we have investigated whether a positive coupling of GABA(B) receptors to adenylyl cyclase could be detected in a cell-free preparation of rat olfactory bulb, a brain region where other Gi/Go-coupled neurotransmitter receptors have been found to stimulate the cyclase activity. The GABA(B) receptor agonist (-)-baclofen significantly increased basal adenylyl cyclase activity in membranes of the granule cell and external plexiform layers, but not in the olfactory nerve-glomerular layer. The adenylyl cyclase stimulation was therefore examined in granule cell layer membranes. The (-)-baclofen stimulation (pD2=4.53) was mimicked by 3-aminopropylphosphinic acid (pD2=4.60) and GABA (pD2=3.56), but not by (+)-baclofen, 3-aminopropylphosphonic acid, muscimol and isoguvacine. The stimulatory effect was counteracted by the GABA(B) receptor antagonists CGP 35348 (pA2=4.31), CGP 55845 A (pA2=7.0) and 2-hydroxysaclofen (pKi=4.22). Phaclofen (1 mM) was inactive. The (-)-baclofen stimulation was not affected by quinacrine, indomethacin, nordihydroguaiaretic acid and staurosporine, but was completely prevented by pertussis toxin and significantly reduced by the alpha subunit of transducin, a betagamma scavenger. The betagamma subunits of transducin stimulated the cyclase activity and this effect was not additive with that produced by (-)-baclofen. In the external plexiform and granule cell layers, but not in the olfactory nerve-glomerular layer, (-)-baclofen enhanced the adenylyl cyclase stimulation elicited by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) 38. Conversely, the adenylyl cyclase activity stimulated by either forskolin or Ca2+/calmodulin-(Ca2+/CaM) was inhibited by (-)-baclofen in all the olfactory bulb layers examined. These data demonstrate that in specific layers of rat olfactory bulb activation of GABA(B) receptors enhances basal and neurotransmitter-stimulated adenylyl cyclase activities by a mechanism involving betagamma subunits of Gi/Go. This positive coupling is associated with a widespread inhibitory effect on forskolin- and Ca2+/CaM-stimulated cyclic AMP formation. (+info)
Signalling by CXC-chemokine receptors 1 and 2 expressed in CHO cells: a comparison of calcium mobilization, inhibition of adenylyl cyclase and stimulation of GTPgammaS binding induced by IL-8 and GROalpha.
The effect of interleukin-8 (IL-8) and growth-related oncogene alpha (GROalpha) on [35S]-guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding, forskolin-stimulated cyclic AMP accumulation and cytosolic calcium concentration were determined in recombinant CHO cells expressing HA-tagged CXC-chemokine receptors 1 and 2 (CXCR1 and CXCR2). Radioligand binding assays confirmed that the binding profiles of the recombinant receptors were similar to those of the native proteins. IL-8 displaced [125I]-IL-8 binding to CXCR1 and CXCR2 with pKi values of 8.89+/-0.05 and 9.27+/-0.03, respectively. GROalpha, a selective CXCR2 ligand, had a pKi value of 9.66+/-0.39 at CXCR2 but a pKi>8 at CXCR1. Calcium mobilization experiments were also consistent with previous reports on native receptors. Activation of both receptors resulted in stimulation of [35S]GTPgammaS binding and inhibition of adenylyl cyclase. A comparison of the functional data at CXCRI showed that a similar potency order (IL-8> >GROalpha) was obtained in all three assays. However, at CXCR2 whilst the potency orders for calcium mobilization and inhibition of adenylyl cyclase were similar (IL-8 > or = GROalpha), the order was reversed for stimulation of [35S]GTPgammaS binding (GROalpha > IL-8). All of the functional responses at both receptors were inhibited by pertussis toxin (PTX), suggesting coupling to a Gi/Go protein. However, the calcium mobilization induced by IL-8 at CXCR1 was not fully inhibited by PTX, suggesting an interaction with a G-protein of the Gq family. Our results with pertussis toxin also suggested that, in the [35S]GTPgammaS binding assay, CXCR1 displays some constitutive activity. Thus, we have characterized the binding and several functional responses at HA-tagged CXCRs 1 and 2 and have shown that their pharmacology agrees well with that of the native receptors. We also have preliminary evidence that CXCR1 displays constitutive activity in our cell line and that CXCR2 may traffic between different PTX sensitive G-proteins. (+info)
Cell-specific coupling of PGE2 to different transduction pathways in arginine vasopressin- and glucagon-sensitive segments of the rat renal tubule.
1. The aim of the present study was to investigate the transduction pathways elicited by prostaglandin E2 (PGE2) to inhibit hormone-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the outer medullary collecting duct (OMCD) and medullary thick ascending limb (MTAL) microdissected from the rat nephron. 2. In the OMCD, 0.3 microM PGE2 and low concentrations of Ca2+ ionophores (10 nM ionomycin or 50 nM A23187) inhibited by about 50% a same pool of arginine vasopressin (AVP)-stimulated cyclic AMP content through a same process insensitive to Bordetella pertussis toxin (PTX). 3. Sulprostone, an agonist of the EP1/EP3 subtypes of the PGE2 receptor, decreased AVP-dependent cyclic AMP accumulation in OMCD and MTAL samples. The concentration eliciting half-maximal inhibition was of about 50 nM in OMCD and 0.1 nM in MTAL. 4. In MTAL, 1 nM sulprostone and PGE2 inhibited by about 90% a same pool of AVP-dependent cyclic AMP content through a PTX-sensitive, Ca2+ -independent pathway. 5. In the OMCD, PGE2 decreased by about 50% glucagon-dependent cyclic AMP synthesis by a process sensitive to PTX and Ca2+ -independent. Sulprostone 1 nM induced the same level of inhibition. 6. These results demonstrate that PGE2 decrease hormone-dependent cyclic AMP accumulation through a G(alpha)i-mediated inhibition of adenylyl cyclase activity in MTAL cells and glucagon-sensitive cells of the OMCD or through a PTX-insensitive increase of intracellular Ca2+ concentration in AVP-sensitive cells of the OMCD. (+info)
The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status.
The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status. (+info)
Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope.
Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity. (+info)
Activation of adenylate cyclase by human recombinant sst5 receptors expressed in CHO-K1 cells and involvement of Galphas proteins.
1. The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO-K1) cells to adenylate cyclase was investigated using receptor selective ligands. 2. Forskolin (10 microM)-stimulated adenosine 3': 5'-cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin-14 and a number of receptor-selective agonists with a rank order of agonist potency typical of the sst5 receptor. L-362,855 and BIM-23056 behaved as full agonists. At higher somatostatin-14 concentrations there was sub-maximal inhibition resulting in a bell-shaped concentration-effect relationship. Pertussis toxin (PTx; 100 ng ml(-1), 18 h) pre-treatment abolished agonist-mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations. 3. The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 microM somatostatin-14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. 4. Pre-treatment with cholera toxin (ChTx; 20 microg ml(-1), 18 h) markedly inhibited sst5 receptor-mediated increases in cyclic AMP formation in intact cells. Somatostatin-14-stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO-K1 membranes and was inhibited by the synthetic peptide Galphasacetyl-354-372-amide (100 microM) by 65.9+/-3.5%, implicating a Galphas protein involvement in this response. 5. Activation of Galphas proteins by somatostatin-14 could be demonstrated with [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding and subsequent immunoprecipitation of 35S labelled Galphas proteins with anti-Galphas serum. 6. These data show that the sst5 receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving Galphas protein activation. (+info)