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Optogenetic Modulation of an Adenylate Cyclase in Toxoplasma gondii Demonstrates a Requirement of the Parasite cAMP for Host-Cell Invasion and Stage Differentiation*

Open AccessPublished:March 22, 2013DOI:https://doi.org/10.1074/jbc.M113.465583
      Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models.

      Introduction

      Toxoplasma gondii is an obligate intracellular parasite of nearly all vertebrates. Other related parasites of medical and veterinary importance include Plasmodium, Eimeria, Cryptosporidium, Neospora, Babesia, and Theileria. Toxoplasma causes ocular and cerebral toxoplasmosis in individuals with immune dysfunction and in developing fetuses and neonates. The parasite also inflicts spontaneous abortions in animals, and thus imposes an economic burden (
      • Torgerson P.R.
      • Macpherson C.N.
      The socioeconomic burden of parasitic zoonoses: global trends.
      ). In addition, T. gondii serves as a widely used model to investigate pathogen-host interactions and protozoan development. Its type I strains exist mainly as a fast replicating tachyzoite stage and cause tissue necrosis (acute infection), while type II strains can also form tissue-dwelling bradyzoite cysts, which persist for the entire life of the host (chronic infection). Successful infection and transmission of Toxoplasma rely on multiplication, persistence, and inter-conversion of these two asexual stages (
      • Sullivan Jr., W.J.
      • Jeffers V.
      Mechanisms of Toxoplasma gondii persistence and latency.
      ).
      The cyclic nucleotides (cAMP and cGMP) are universal regulators of cell signaling. They are generated from ATP or GTP by the catalytic action of adenylate cyclase or guanylate cyclase, respectively. The adenylate cyclases involved in cellular signaling belong to class III; some are membrane-bound, and others are cytosolic in metazoans (
      • Linder J.U.
      Class III adenylyl cyclases: molecular mechanisms of catalysis and regulation.
      ). The membrane-bound isoforms are commonly regulated by G-proteins in response to external stimuli, whereas the soluble ones respond to intracellular signals, such as calcium and bicarbonate levels (
      • Linder J.U.
      Class III adenylyl cyclases: molecular mechanisms of catalysis and regulation.
      ). The most prominent examples of cAMP-activated proteins include protein kinase A (PKA), transcription factors (e.g. CREB-1), and cAMP-gated ion channels (
      • Vandamme J.
      • Castermans D.
      • Thevelein J.M.
      Molecular mechanisms of feedback inhibition of protein kinase A on intracellular cAMP accumulation.
      ,
      • Biel M.
      • Wahl-Schott C.
      • Michalakis S.
      • Zong X.
      Hyperpolarization-activated cation channels: from genes to function.
      ,
      • Altarejos J.Y.
      • Montminy M.
      CREB and the CRTC co-activators: sensors for hormonal and metabolic signals.
      ). Upon activation, PKA, for instance, can phosphorylate its target proteins, and exerts numerous effects, including gene modulation and ion conductance. Phosphodiesterases degrade cAMP to counter-regulate its cellular level, which is strictly controlled (
      • Vandamme J.
      • Castermans D.
      • Thevelein J.M.
      Molecular mechanisms of feedback inhibition of protein kinase A on intracellular cAMP accumulation.
      ,
      • Biel M.
      • Wahl-Schott C.
      • Michalakis S.
      • Zong X.
      Hyperpolarization-activated cation channels: from genes to function.
      ,
      • Altarejos J.Y.
      • Montminy M.
      CREB and the CRTC co-activators: sensors for hormonal and metabolic signals.
      ,
      • Conti M.
      • Beavo J.
      Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling.
      ,
      • Dao K.K.
      • Teigen K.
      • Kopperud R.
      • Hodneland E.
      • Schwede F.
      • Christensen A.E.
      • Martinez A.
      • Døskeland S.O.
      Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition.
      ). The affinities of cAMP signaling-associated proteins such as of PKA and phosphodiesterase for cAMP range from nanomolar to micromolar amounts. It has been shown that an activator of adenylate cyclase, forskolin, can exert a transient rise in cAMP levels of Toxoplasma-infected cells and induces bradyzoite formation (
      • Kirkman L.A.
      • Weiss L.M.
      • Kim K.
      Cyclic nucleotide signaling in Toxoplasma gondii bradyzoite differentiation.
      ,
      • Eaton M.S.
      • Weiss L.M.
      • Kim K.
      Cyclic nucleotide kinases and tachyzoite-bradyzoite transition in Toxoplasma gondii.
      ). In contrast, a membrane-permeable and nonhydrolysable analog of cAMP as well as phosphodiesterase inhibitors can cause a persistent activation of cAMP-dependent host and/or parasite pathways resulting in a reduced bradyzoite differentiation (
      • Kirkman L.A.
      • Weiss L.M.
      • Kim K.
      Cyclic nucleotide signaling in Toxoplasma gondii bradyzoite differentiation.
      ,
      • Eaton M.S.
      • Weiss L.M.
      • Kim K.
      Cyclic nucleotide kinases and tachyzoite-bradyzoite transition in Toxoplasma gondii.
      ). It is, however, debatable whether the parasite or host cAMP determines the stage switching in T. gondii. Other roles of cAMP described in Plasmodium, e.g. host-cell invasion (
      • Ono T.
      • Cabrita-Santos L.
      • Leitao R.
      • Bettiol E.
      • Purcell L.A.
      • Diaz-Pulido O.
      • Andrews L.B.
      • Tadakuma T.
      • Bhanot P.
      • Mota M.M.
      • Rodriguez A.
      Adenylyl cyclase α and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.
      ,
      • Leykauf K.
      • Treeck M.
      • Gilson P.R.
      • Nebl T.
      • Braulke T.
      • Cowman A.F.
      • Gilberger T.W.
      • Crabb B.S.
      Protein kinase A-dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite.
      ), also remain to be identified in T. gondii.
      Typically, the study of cAMP-mediated pathways depends on chemical activators and inhibitors affecting protein kinases and phosphodiesterases, as well as on membrane-permeable analogs of cyclic nucleotides. The use of such commercial modulators, targeting primarily the mammalian proteins, is not appropriate to examine cAMP signaling in intracellular pathogens. A concomitant and pleiotropic action of these drugs on the host cell and on the enclosed pathogen often obscures the interpretation of results. Unambiguous and comprehensive research on cAMP signaling in two-organism systems, such as pathogen-infected host cells or in symbiotic models, therefore requires specific, efficient, and spatiotemporal control of the cellular cAMP levels within individual partners. Noninvasive control of biological processes by photoactivated molecules has become a common method in recent years (
      • Deisseroth K.
      Optogenetics.
      ). Optogenetic tools have gained significant momentum following the discovery of channelrhodopsin that allows modulation of the membrane voltage, a general parameter applicable to essentially all cells (
      • Nagel G.
      • Ollig D.
      • Fuhrmann M.
      • Kateriya S.
      • Musti A.M.
      • Bamberg E.
      • Hegemann P.
      Channelrhodopsin-1: a light-gated proton channel in green algae.
      ). Its wide application has stimulated the demand for the photoregulatable proteins to modulate general regulators such as cyclic nucleotides and inositides (
      • Schröder-Lang S.
      • Schwärzel M.
      • Seifert R.
      • Strünker T.
      • Kateriya S.
      • Looser J.
      • Watanabe M.
      • Kaupp U.B.
      • Hegemann P.
      • Nagel G.
      Fast manipulation of cellular cAMP level by light in vivo.
      ,
      • Idevall-Hagren O.
      • Dickson E.J.
      • Hille B.
      • Toomre D.K.
      • De Camilli P.
      Optogenetic control of phosphoinositide metabolism.
      ). Recently, a photoregulatable and soluble adenylate cyclase from the lithotropic bacterium Beggiatoa has been reported (
      • Ryu M.H.
      • Moskvin O.V.
      • Siltberg-Liberles J.
      • Gomelsky M.
      Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications.
      ,
      • Stierl M.
      • Stumpf P.
      • Udwari D.
      • Gueta R.
      • Hagedorn R.
      • Losi A.
      • Gärtner W.
      • Petereit L.
      • Efetova M.
      • Schwarzel M.
      • Oertner T.G.
      • Nagel G.
      • Hegemann P.
      Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa.
      ). We utilized this bacterial adenylate cyclase to recognize the roles of cAMP in the asexual stages of T. gondii, as well as to establish its general application in a model pathogen.

      DISCUSSION

      We have generated transgenic Toxoplasma strains expressing a photoactivated adenylate cyclase from a lithotropic bacterium. The enzyme is cytosolic and exerts a notable induction of the parasite cAMP level. A hierarchical control of cAMP by enzyme expression and photoactivation offers a flexible modulation of cyclic nucleotide in diverse setups, such as during the infective-extracellular or replicative-intracellular stages. In addition a conditional expression of bPAC in type I and II strains allows analysis of cAMP-mediated signaling in acute and dormant stages of T. gondii. The advantages of the new method are as follows: (a) specific and quantitative cAMP regulation within the parasite or host cell; (b) bPAC is gene-encoded, i.e. inheritable to the progeny; (c) it circumvents routine problems in culture, e.g. poor diffusion, premature degradation, and sustained activation when using chemical modulators of cAMP; (d) a transient, persistent, and reversible control of cAMP can be achieved to satisfy versatile experimental setups; (e) a functional bPAC can be expressed irrespective of the N- and/or C-terminal fusion, or epitope size (GFP, mCherry, Myc, and ddFKBP), and is thus suitable for a spatial control of different cAMP pools by organelle-specific targeting. Although useful for a modest cAMP induction, the dark activity of bPAC may pose a drawback (this work and Ref.
      • Stierl M.
      • Stumpf P.
      • Udwari D.
      • Gueta R.
      • Hagedorn R.
      • Losi A.
      • Gärtner W.
      • Petereit L.
      • Efetova M.
      • Schwarzel M.
      • Oertner T.G.
      • Nagel G.
      • Hegemann P.
      Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa.
      ). This problem can however be resolved by conditional expression, which also avoids the undesired expression of a foreign protein in routine cultures. A much higher and more dynamic cAMP induction further advocates the use of a conditional expression in our model. We are now extending the optogenetic application to a mutagenized isoform of bPAC for regulating the cGMP levels in T. gondii (
      • Ryu M.H.
      • Moskvin O.V.
      • Siltberg-Liberles J.
      • Gomelsky M.
      Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications.
      ). Quite notably, this method can also be applied to other gene-tractable intertwined models, for instance to study symbiotic and pathogen-host interactions/relationships.
      Cyclic nucleotide-mediated signaling is known to regulate multiple processes in other pathogens, including the protozoan parasites (
      • Gould M.K.
      • de Koning H.P.
      Cyclic-nucleotide signaling in protozoa.
      ,
      • McDonough K.A.
      • Rodriguez A.
      The myriad roles of cyclic AMP in microbial pathogens: from signal to sword.
      ). Much of our knowledge in apicomplexan parasites is derived from Plasmodium species, where cAMP signaling is required for exocytosis, host-cell invasion, and gametocyte differentiation (
      • Ono T.
      • Cabrita-Santos L.
      • Leitao R.
      • Bettiol E.
      • Purcell L.A.
      • Diaz-Pulido O.
      • Andrews L.B.
      • Tadakuma T.
      • Bhanot P.
      • Mota M.M.
      • Rodriguez A.
      Adenylyl cyclase α and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.
      ,
      • Leykauf K.
      • Treeck M.
      • Gilson P.R.
      • Nebl T.
      • Braulke T.
      • Cowman A.F.
      • Gilberger T.W.
      • Crabb B.S.
      Protein kinase A-dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite.
      ,
      • Baker D.A.
      Cyclic nucleotide signalling in malaria parasites.
      ). Consistent with Plasmodium, our results suggest a role of cAMP in host-cell invasion by Toxoplasma tachyzoites that remains to be ascertained by parasite secretion studies. Interestingly, a moderate induction of cAMP (achieved by Shield1) appears to be sufficient for regulating the invasion/secretion events, as a light exposure did not increase the observed invasion defect in type I tachyzoites. In contrast, a much higher threshold of cAMP was required to induce bradyzoite formation in type II parasites, which could only be achieved by concurrent protein stabilization and photoactivation. It is well established that physicochemical (pH, temperature, sodium nitroprusside etc.) stress can induce tachyzoite-to-bradyzoite conversion (this work and Refs.
      • Sullivan Jr., W.J.
      • Jeffers V.
      Mechanisms of Toxoplasma gondii persistence and latency.
      ,
      • Ferreira da Silva Mda F.
      • Barbosa H.S.
      • Gross U.
      • Lüder C.G.
      Stress-related and spontaneous stage differentiation of Toxoplasma gondii.
      ) and that cyclic nucleotides are general mediators of stress signaling and differentiation in many other microbes (
      • Gould M.K.
      • de Koning H.P.
      Cyclic-nucleotide signaling in protozoa.
      ,
      • McDonough K.A.
      • Rodriguez A.
      The myriad roles of cyclic AMP in microbial pathogens: from signal to sword.
      ). Our work illustrates the importance of the parasite cAMP for its differentiation and suggests a conserved cAMP-mediated stress signaling in T. gondii. Consistently, the parasite encodes all imperative factors required for a generic cAMP signaling, e.g. adenylate cyclases, phosphodiesterases, protein kinases, regulatory subunits, and cAMP-binding proteins (Toxoplasma database). In other models, the PKA-dependent as well as PKA-independent pathways can control gene expression (
      • Sands W.A.
      • Palmer T.M.
      Regulating gene transcription in response to cyclic AMP elevation.
      ). The availability of the bPAC-expressing optogenetic strains allows a dissection of such downstream events in T. gondii.
      Bradyzoites and tachyzoites differ in their stress tolerance, metabolism and growth rates, which depend on a rewiring of gene expression (
      • Sullivan Jr., W.J.
      • Jeffers V.
      Mechanisms of Toxoplasma gondii persistence and latency.
      ,
      • Ferreira da Silva Mda F.
      • Barbosa H.S.
      • Gross U.
      • Lüder C.G.
      Stress-related and spontaneous stage differentiation of Toxoplasma gondii.
      ). Tachyzoites express a unique set of proteins, including Sag1, Eno2 and Ldh1, whereas bradyzoites show a distinct expression of other proteins, e.g. Cst1, Bag1, Eno1 and Ldh2 (
      • Radke J.R.
      • Behnke M.S.
      • Mackey A.J.
      • Radke J.B.
      • Roos D.S.
      • White M.W.
      The transcriptome of Toxoplasma gondii.
      ,
      • Cleary M.D.
      • Singh U.
      • Blader I.J.
      • Brewer J.L.
      • Boothroyd J.C.
      Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression.
      ). Using our method, we reveal the requirement of the parasite cAMP for stage-specific expression (pLDH2, pCST1, and pSAG1). A family of cAMP-responsive factors controls transcription by binding to the cAMP-response elements in other eukaryotes (
      • Sands W.A.
      • Palmer T.M.
      Regulating gene transcription in response to cyclic AMP elevation.
      ). Although it is known that transcription of the bradyzoite genes is directed by autonomous promoter elements (
      • Behnke M.S.
      • Radke J.B.
      • Smith A.T.
      • Sullivan Jr., W.J.
      • White M.W.
      The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements.
      ), their binding factors remain to be characterized. In this regard, the plant-like AP2 transcription factors are currently the most promising candidates (
      • Balaji S.
      • Babu M.M.
      • Iyer L.M.
      • Aravind L.
      Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains.
      ). One such AP2 factor has recently been shown to govern the stage-specific expression and differentiation (
      • Walker R.
      • Gissot M.
      • Croken M.M.
      • Huot L.
      • Hot D.
      • Kim K.
      • Tomavo S.
      The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation.
      ). The activity of AP2 factors is likely regulated by phosphorylation (
      • Treeck M.
      • Sanders J.L.
      • Elias J.E.
      • Boothroyd J.C.
      The phosphoproteomes of Plasmodium falciparumToxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries.
      ) that may be controlled by cAMP signaling. Notably, our optogenetic strain can be utilized to examine the cAMP-mediated epigenetic coding and gene modulations during/upon stage differentiation, as well as to identify the mediators of cAMP signaling. For instance, genome-wide analyses of the gene expression and histone codes in response to low/high cAMP levels could be performed to identify a common set of stage-specific and cAMP-regulated genes. In addition, a follow-up promoter mapping of such genes could be used to classify the signature elements and their binding factors. Along the line, future research should also reveal how a transient or persistent cAMP induction exerts a reciprocal effect on parasite differentiation, which may depend on differential activation of transcriptional regulators.

      Acknowledgments

      We thank Manuela Stierl for providing bPAC ORF in pET28a and pCMV plasmids. We are grateful to Jörn van Buer (Humboldt University, Berlin) and Suneel Kateriya (Delhi University, India) for initial contributions.

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