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Bacterial effector binds host cell adenylyl cyclase to potentiate G?s-dependent cAMP production.

ABSTRACT: Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the ?-subunit of the AC-stimulating G protein (G?s) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of G?s and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and G?s; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the G?s-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein ?-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released G?s. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.

SUBMITTER: Pulliainen AT

PROVIDER: S-EPMC3386119 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production.

Pulliainen Arto T AT Pieles Kathrin K Brand Cameron S CS Hauert Barbara B Böhm Alex A Quebatte Maxime M Wepf Alexander A Gstaiger Matthias M Aebersold Ruedi R Dessauer Carmen W CW Dehio Christoph C

Proceedings of the National Academy of Sciences of the United States of America 20120525 24

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics ...[more]

PMID: 22635269

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Project description:Background & aimsGenetic defects in polycystin-1 or -2 (PC1 or PC2) cause polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway, leading to increased vascular endothelial growth factor A (VEGF-A) signaling. In PC2-defective cholangiocytes, cAMP production in response to [Ca2+]ER depletion is increased, while store-operated Ca2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca2+]ER levels are reduced. We investigated whether the adenylyl cyclases, AC5 and AC6, which can be inhibited by Ca2+, are activated by the ER chaperone STIM1. This would result in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation in PC2-defective cells, in response to [Ca2+]ER depletion.MethodsPC2/AC6 double conditional knockout (KO) mice were generated (Pkd2/AC6 KO) and compared to Pkd2 KO mice. The AC5 inhibitor SQ22,536 or AC5 siRNA were used in isolated cholangiocytes while the inhibitor was used in biliary organoid and animals; liver tissues were harvested for histochemical analysis.ResultsWhen comparing Pkd2/AC6 KO to Pkd2 KO mice, no decrease in liver cyst size was found, and cellular cAMP after [Ca2+]ER depletion only decreased by 12%. Conversely, in PC2-defective cells, inhibition of AC5 significantly reduced cAMP production, pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced growth of biliary organoids derived from Pkd2 KO and Pkd2/AC6 KO mice. In vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. After [Ca2+]ER depletion in PC2-defective cells, STIM1 interacts with AC5 but not with Orai1, the Ca2+ channel that mediates SOCE.Conclusion[Ca2+]ER depletion in PC2-defective cells activates AC5 and results in stimulation of cAMP/ERK1-2 signaling, VEGF production and cyst growth. This mechanism may represent a novel therapeutic target.Lay summaryPolycystic liver diseases are characterized by progressive cyst growth until their complications mandate surgery or liver transplantation. In this manuscript, we demonstrate that inhibiting cell proliferation, which is induced by increased levels of cAMP, may represent a novel therapeutic target to slow the progression of the disease.

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Bacterial effector binds host cell adenylyl cyclase to potentiate G?s-dependent cAMP production.

Publications

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production.

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