The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem(GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

Autoinhibitory mechanism and activity-related structural changes in a mycobacterial adenylyl cyclase

An adenylyl cyclase from Mycobacterium avium, Mal 120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Mal 120 in a monomeric form and its truncated construct as a dimer. Mal 120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional alpha-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. (C) 2015 Elsevier Inc. All rights reserved.

New structural forms of a mycobacterial adenylyl cyclase Rv1625c

Rv1625c is one of 16 adenylyl cyclases encoded in the genome of Mycobacterium tuberculosis. In solution Rv1625c exists predominantly as a monomer, with a small amount of dimer. It has been shown previously that the monomer is active and the dimeric fraction is inactive. Both fractions of wild-type Rv1625c crystallized as head-to-head inactive domain-swapped dimers as opposed to the head-to-tail dimer seen in other functional adenylyl cyclases. About half of the molecule is involved in extensive domain swapping. The strain created by a serine residue located on a hinge loop and the crystallization condition might have led to this unusual domain swapping. The inactivity of the dimeric form of Rv1625c could be explained by the absence of the required catalytic site in the swapped dimer. A single mutant of the enzyme was also generated by changing a phenylalanine predicted to occur at the functional dimer interface to an arginine. This single mutant exists as a dimer in solution but crystallized as a monomer. Analysis of the structure showed that a salt bridge formed between a glutamate residue in the N-terminal segment and the mutated arginine residue hinders dimer formation by pulling the N-terminal region towards the dimer interface. Both structures reported here show a change in the dimerization-arm region which is involved in formation of the functional dimer. It is concluded that the dimerization arm along with other structural elements such as the N-terminal region and certain loops are vital for determining the oligomeric nature of the enzyme, which in turn dictates its activity.

Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

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Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC''. The calpain-mediated ACT processing allows trafficking of the "soluble AC'' domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools'', which would play different roles in the cell pathophysiology.

Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel Deuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.

Pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) which belongs to the secretin/glucagon/ VIP family has been originally isolated from the sheep hypothalamus on the basis of its ability to stimulate cAMP formation in culture rat anterior pituitary cells. Post-translational processing of the PACAP precursor generates two biologically active molecular forms, PACAP-38 and PACAP-27. The primary structure of PACAP has been remarkably conserved during evolution. The sequence of PACAP-27 exhibits substantial similarities with those of vasoactive intestinal polypeptide (VIP), glucagon and secretin. The gene encoding the PACAP precursor is widely expressed in brain and various peripheral organs, notably in endocrine glands, gastro-intestinal, urogenital tracts and respiratory system. In vivo, and in vitro studies have shown that PACAP exhibits multiple activities especially a trophic activity during ontogenesis, notably in the adrenal medulla and the central nervous system. The biological effects of PACAP are mediated through three distinct receptor subtypes which exhibit differential affinities for PACAP and VIP. The PAC1 receptor, which shows high selectivity for PACAP, is coupled to several transduction systems. In contrast, VPAC1 and VPAC2, which bind with the same affinity for PACAP and VIP, are mainly coupled to the adenylyl cyclase pathway. In conclusion, PACAP is neuropeptide, and it functions as a hypothalamic hormone, neurohormone, neuromodulator, vasodilator, neurotransmitter or trophic factor in the brain and the various organs.

Lineage-specific domain fusion in the evolution of purine nucleotide cyclases in cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.