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Infection and Immunity, October 2008, p. 4757-4763, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00527-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Role of Protein Kinase A in Trypanosoma cruzi ^,

Yi Bao,¹ Louis M. Weiss,^1,2 Vicki L. Braunstein,¹ and Huan Huang^1*

Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461,¹ Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461²

Received 29 April 2008/ Returned for modification 27 May 2008/ Accepted 3 August 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Protein kinase A (PKA) is an important mediator of many signal transduction pathways that occur in eukaryotic cells, and it has been implicated as a regulator of stage differentiation in Trypanosoma cruzi. To evaluate the importance of the PKA catalytic subunit of T. cruzi (TcPKAc), a gene encoding a PKA inhibitor (PKI) containing a specific PKA pseudosubstrate, R-R-N-A, was subcloned into a pTREX vector and introduced into epimastigotes by electroporation. Expression of PKI has a lethal effect in this parasite. Similarly, a pharmacological inhibitor, H89, killed epimastigotes at a concentration of 10 µM. To understand the biology of PKA, identification of the particular substrates of this enzyme is essential. Using a yeast two-hybrid system, 38 candidates interacting with TcPKAc were identified. Eighteen of these were hypothetical proteins with unknown functions, while the others had putative or known functions. The entire open reading frames of eight genes presumably important in regulating T. cruzi growth, adaptation, and differentiation, including a type III PI3 kinase (Vps34), a putative PI3 kinase, a putative mitogen-activated extracellular signal-regulated kinase, a cyclic AMP (cAMP)-specific phosphodiesterase (PDEC2), a hexokinase, a putative ATPase, a DNA excision repair protein, and an aquaporin were confirmed to interact with TcPKAc in the yeast Saccharomyces cerevisiae under the highest stringency selection conditions, and PKA phosphorylated the recombinant proteins of these genes. Taken together, these findings demonstrate the importance of cAMP-PKA signaling in this organism.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Trypanosoma cruzi causes Chagas’ disease, a chronic debilitating disease in Central and South America. This disease is also recognized as an opportunistic infection in immunocompromised patients with acquired immunodeficiency syndrome (6, 14, 15, 23, 26, 27, 34). Currently, the available therapeutic agents for T. cruzi are highly toxic, and there is no effective treatment for chronic Chagas’ disease. Understanding the basic biology of this organism is essential for developing new targets for treatment. Protein kinase A (PKA) has been implicated in stage differentiation in T. cruzi (9, 18). We have previously reported the molecular cloning and characterization of both the PKA catalytic subunit of T. cruzi (TcPKAc) and the PKA regulatory subunit of T. cruzi (TcPKAr) (12, 13).

PKA regulates fundamental pathways in many organisms. It has been extensively studied and is one of the best-known members of the PK family (28, 31). In Saccharomyces cerevisiae, PKA regulates cell growth by conveying signals from the small GTP-binding Ras proteins (2, 10, 29). The two Ras proteins, Ras1 and Ras2, increase adenylyl cyclase activity and stimulate the production of cAMP (7, 30). This stimulation results in elevated PKA activity and the increased phosphorylation of substrates that are important for cell growth and proliferation (32). In response to starvation, Dictyostelium discoideum becomes a multicellular organism. cAMP acts as both an extracellular messenger, which is secreted by cells in response to starvation, and an intracellular activator of PKA. Components of the cAMP pathway, including PKA, are essential for differentiation of the cellular components of the fruiting body (16). In African trypanosomes, cAMP induces the cell cycle arrest that occurs in the differentiation of bloodstream trypanosomes to procylic forms (33). In Plasmodium species that cause malaria, cAMP and levels of PKA have been implicated in gametocyte differentiation (21).

PKA catalyzes the transfer of the -position phosphate from ATP to the hydroxyl group of particular serine or threonine residues in its substrates. This phosphorylation ultimately alters cell physiology by modifying the activities associated with these substrate proteins. In this paper, we report that the blockade of PKA function by a genetic approach or a PKA-specific inhibitor, H89, results in a lethal effect for T. cruzi, indicating that PKA activity is critical for the viability of T. cruzi. To understand the biology of PKA, identification of the particular substrates of this enzyme is essential. Using a yeast two-hybrid system, 38 candidates interacting with TcPKAc were identified. Among the identified genes, we have further confirmed eight candidates presumably important in regulating T. cruzi growth and differentiation as the substrates of TcPKAc. The identification of these substrates enables us to assess the functions of cAMP-PKA signaling in T. cruzi.

We have previously published on the similarities and differences between TcPKAc and mammalian PKA (13). TcPKAc or the PKA regulatory subunit of T. cruzi can potentially serve as a drug target. For the design of inhibitors, several approaches can be utilized, including inhibitors that target the ATP binding pocket and substrate tethering sites for the catalytic subunit, inhibitors that target the activation of the kinase, or inhibitors that disrupt targeting. An additional strategy is to target the synthesis and degradation of the second messenger, cAMP. We believe that disrupting the interactions between TcPKAc and selected downstream interacting proteins could be a unique and potentially selective approach if we are able to identify critical interacting proteins in this pathway in this pathogen.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell culture. Trypanosoma cruzi epimastigotes (HO 3/15, Brazil, Tulahuen, and CL Brener) were grown at 26°C in liver infusion tryptose (LIT) broth supplemented with 10% fetal calf serum (Gibco Life Technologies, Gaithersburg, MD).

Blockade of PKAc in T. cruzi. Reverse transcription-PCR (RT-PCR) was used to amplify a PKI peptide (GenBank accession number BC026550) tagged with a hemagglutin (HA) epitope at the C terminus from mouse muscle total RNA with primers (Table 1). PKI-HA was subcloned into a pTREX vector and the pTREX-PKI-HA construct was introduced into epimastigotes (the Tulahuen strain) by electroporation. Briefly, epimastigotes in the late logarithmic growth phase in LIT broth were collected and washed with phosphate-buffered saline (PBS) I buffer (132 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2) by centrifuging at 1,200 x g at room temperature. The parasite pellet was resuspended in PBS II buffer (PBS I buffer plus 0.5 mM MgOAc₂, 0.1 mM CaCl₂) at a final density of 1.4 x 10⁸ to 2.0 x 10⁸ cells/ml, and 375 µl of the parasite suspension was incubated with 100 µg pTREX-PKI-HA construct DNA and adjusted to 400 µl of the final volume. Electroporation was performed in a BTX disposable cuvette using an Electro cell manipulator (BTX Genetronics, Inc., San Diego, CA) with one pulse delivered to the parasites in a setting of 375 V, 25 , and 50 µF. Subsequently, the transfected parasite suspension was diluted with 10 ml of LIT medium and incubated for 48 h at 26°C. G418 was then added to the LIT containing the transfected parasites at a final concentration of 100 µg/ml and then increased to 250 µg/ml or 500 µg/ml. Two control conditions were performed for this transfection. An equal amount of empty vector DNA (pTREX) or an equal volume of Tris-EDTA (TE) buffer without the construct DNA (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) was added to the parasite suspension, and the same procedure of electroporation and G418 selection was followed as mentioned above. As a complementary approach, H89 (Sigma, St. Louis, MO) was used for another PKA blockade study (5). The drug was added to epimastigote cultures at various concentrations from 5 to 20 µM and incubated using the standard culture conditions described above.

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TABLE 1. PCR primers for candidate gene products

Construction of a GAL4 AD library and performance of a yeast two-hybrid screen. T. cruzi (the HO 3/15 strain) genomic DNA was obtained following the protocol of the Wizard genomic DNA purification kit with modification (Promega, Madison, WI). Briefly, 4 x 10⁹ epimastigotes were pelleted and washed with 1x PBS by centrifugation. The pellet was resuspended in 300 µl TE buffer and subjected to three freeze-thaw cycles. Afterwards, the nuclei were solubilized by adding 300 µl of nuclei lysis solution to the freeze-thaw lysate. Proteins were then precipitated by adding 100 µl of protein precipitation solution, followed by centrifugation at 16,000 x g for 3 minutes. DNA was then precipitated by isopropanol, washed by 70% ethanol, and subsequently rehydrated. RNase was added to the DNA sample, and the sample was incubated at 37°C for 30 min and then incubated 65°C for 1 h. Genomic DNA was stored at 4°C. The construction of the GAL4 AD library was carried out by using the method of the HybriZAP-2.1 XR library construction kit (Stratagene, La Jolla, CA). Briefly, genomic DNA was digested with restriction enzymes (XhoI and EcoRI) and then electrophoresed on a 0.7% agarose gel. DNA was purified from the region of the gel containing digested DNA between 2 and 9 kb, ligated into the HybriZAP-2.1 vector, and then packaged using high-efficiency Gigapack III Gold packaging extract. The titers of the library were determined, and the library was subsequently amplified. A pAD-GAL4-2.1 phagemid library was produced by mass excision using the eXassist helper phage according to the manufacturer's instructions (Stratagene, La Jolla, CA). The bait construct (using the binding domain [BD] of GAL4) was produced by ligating the full-length open reading frame (ORF) of TcPKAc (13) (GenBank accession number AY055783) with EcoRI and PstI restriction sites into pBD plasmids to generate pBD-TcPKAc. Large-scale transformation of the bait construct pBD-TcPKAc with the AD plasmid library was carried out using YRG-2 Saccharomyces cerevisiae yeast competent cells under a high-stringency screen according to the manufacturer's protocol (Stratagene, La Jolla, CA). Briefly, 100 µl of competent yeast cells, 100 µg of herring sperm DNA, 10 µg of bait construct pBD-TcPKAc DNA, and 50 µg of AD plasmid library DNA were added into a microcentrifuge tube and gently mixed. Following this, 600 µl of TE-LiAc-polyethylene glycol solution was added, the solution was mixed by vortexing, the tube was incubated at 30°C for 30 min with shaking at 200 rpm, and then 70 µl of dimethyl sulfoxide were added. The samples were then subjected to heat shock at 42°C for 15 min and placed on ice for 10 min. Yeast cells were pelleted by centrifugation at 3,000 rpm for 10 s, resuspended in 0.5 ml TE buffer, and plated onto 150-mm SD-selective plates (250 µl on each plate) that did not contain leucine, tryptophan, and histidine. The plates were incubated at 30°C for 2 to 4 days until colonies appeared. Constructs, pADwt with pBDwt, were used as the positive control, and pADwt with pLaminC was used as the negative control. Each of the colonies from the pBD-TcPKAc and AD library interaction screen were further inoculated into SD medium lacking leucine, tryptophan, and histidine, grew at 30°C for 2 to 4 days, and were then pelleted by centrifugation at 2,000 rpm for 5 min. Afterwards, DNA was extracted using a phenol-chloroform standard method. These plasmids, which contained interacting protein genes, were then transformed into Escherichia coli XL blue (Stratagene, La Jolla, CA) and plated on both LB ampicillin plates and LB chloramphenicol plates. When colonies grew both on ampicillin and on chloramphenicol LB plates (an indication that the colony contained both pAD-Gal4 and pBD-Gal4 constructs), a single colony on LB ampicillin plates (containing the pAD-Gal4-prey gene) was further inoculated in LB ampicillin broth. Subsequently, the plasmid was isolated, and the prey gene was sequenced. Information from prey gene sequences was analyzed using BLAST (GenBank; NCBI).

Among the identified genes, eight candidates were selected, and seven entire ORFs of these genes were then amplified by RT-PCR using the total RNA of CL Brener strain epimastigotes and primers containing appropriate restriction sites (Table 1). Briefly, cDNA was generated using the SuperScript first-strand synthesis system for RT-PCR according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Three micrograms of total RNA was converted into cDNA by incubating with 50 U of SuperScript II reverse transcriptase, oligo(dT), deoxynucleoside triphosphate, and appropriate buffer at 42°C for 50 min. RNase H was then added to degrade the remaining RNA, and the cDNA was stored as a 2-µl aliquot in each PCR tube at –80°C until used. PCRs were performed by adding 2 µl of this cDNA and 3 µl of primer mixes (150 pmol of each primer) into 45 µl of PCR SuperMix High Fidelity solution (Invitrogen, Carlsbad, CA). PCR was performed using the following conditions: initialization at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing (primer dependence) (Table 1), and extension at 72°C for 3 min. A final elongation at 72°C for 10 min followed the 35 cycles, and the mixtures were stored at 4°C until use. The amplicons were purified and digested with appropriate restriction enzymes (Table 1), ligated into pAD-Gal4 vectors, and sequenced to verify the correct genes. In addition, the entire coding region of PDEC2 was obtained from a TcrPDEC2-pET22b+ expression vector (a kind gift from A. C. Schoijet) by digesting the construct with BamHI and XhoI, and then the PDEC2 gene was subcloned into the pAD-Gal4 vector. Repeat transformation using these constructs and the TcPKAc bait construct was performed under the highest stringency conditions.

Recombinant protein expression and purification. Six genes (those for PI3 kinase, extracellular signal-regulated kinase [ERK], hexokinase, ATPase, DNA excision repair protein [DERP], and aquaporin [AQP]) were reamplified by PCR using primers with appropriate restriction sites (Table 2) and subcloned into a pTrcHisA expression vector (Invitrogen, Carlsbad, CA), which expresses six histidines in the N terminus of recombinant proteins. TcrPDEC2-pET22b+ and TcVps34-pDEST (a kind gift from A. C. Schoijet) are also expression constructs producing six histidines in the N terminus of the recombinant proteins of PDEC2 and Vps34. Each of these expression constructs was introduced into E. coli BL21(DE3) (Invitrogen, Carlsbad, CA) by transformation and plated in LB ampicillin agar plates. One colony was inoculated in 5 ml of LB broth with 100 µg ampicillin/ml, grown overnight, and inoculated into 50 ml of LB broth with 100 µg ampicillin/ml. The bacteria were allowed to grow to a density of 0.6 to 0.8 A₆₀₀ with constant agitation at 250 rpm at 37°C and were then induced to express the recombinant protein by adding 0.5 mM isopropylthiogalactoside (Sigma, St. Louis, MO). The induced bacteria were maintained with the same agitation at 28°C for 12 to 16 h. Cells were then harvested and stored at –80°C until needed. Protein purification was performed using the HisTrap FF crude kit (GE Healthcare, Fairfield, CT). Briefly, a 1.5-g bacterial pellet was resuspended in 5 ml of lysis buffer (0.5% Triton X-100, 0.2 mg/ml lysozyme, 1 mM MgCl₂, 20 µg/ml DNase, and one protease inhibitor cocktail tablet per 10 ml of buffer) and incubated on ice for 30 min. Afterwards, 5 ml of binding buffer (1x phosphate buffer with 20 mM imidazole) was added to the lysate, followed by room temperature incubation for 20 min with constant agitation and centrifugation at 27,000 x g at 4°C for 30 min. The supernatant was then passed into a 1-ml HisTrap FF crude column (precharged with Ni²⁺), the column was washed with 10 ml of binding buffer, and the protein was eluted using 5 ml of elution buffer (1x phosphate buffer containing 40 mM imidazole) by collecting the eluate in 1-ml fractions. Purified proteins were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under reducing conditions and stained with Coomassie blue R250. Immunoblotting was then performed using a six-histidine monoclonal antibody (MAb) (Novagen, Darmstadt, Germany) to verify the expression of recombinant proteins. A TcAQP antibody (a kind gift from R. Docampo) was also used to confirm the expression of recombinant TcAQP. The eluate containing the recombinant proteins was dialyzed against 2 liters of Tris buffer (10 mM Tris-HCl, pH 7.4) at 4°C for 24 h, and the protein concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) prior to in vitro phosphorylation assays.

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TABLE 2. PCR primers for expression constructs

Immunoprecipitation and immunoblotting. Using a TcPKAc MAb and a TcAQP antibody, we performed coimmunoprecipitation in T. cruzi protein extract. To produce parasite lysates for coimmunoprecipitation, 6 x 10⁹ CL Brener strain epimastigotes derived from cell culture were resuspended in buffer A (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 1% Triton X-100, and one tablet of protease inhibitor cocktail tablet [Roche, Bavaria, Germany] per 5 ml of buffer) and subjected to three freeze-thaw cycles, followed by centrifugation at 16,000 x g for 30 min at 4°C. The supernatant was then used for immunoprecipitation, followed by immunoblotting. Briefly, 2 mg of Triton X-100 protein extract from epimastigotes was used for each precipitation. A TcPKAc MAb was added at optimal dilutions (1:100) and incubated at 4°C overnight with a rocking motion. An unrelated MAb (anti-BAG5 of Toxoplasma gondii) was used as a negative control. The antibody-antigen complexes were isolated by incubating the reaction mixture with a slurry of protein A-Sepharose CL-4B (Sigma, St. Louis, MO), followed by centrifugation at 10,000 x g and washing three times with buffer A prior to immunoblotting. For immunoblotting, the protein A-Sepharose beads of each immunoprecipitation were resuspended in 30 µl of sample buffer, boiled for 5 minutes, briefly centrifuged, run on a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membrane, blocked with 5% nonfat milk, detected with appropriate primary antibody (a 1:1,000 dilution) and secondary antibody conjugated with alkaline phosphatase (1:5,000 dilution), and visualized by using 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium as a substrate (Roche, Bavaria, Germany).

In vitro phosphorylation. For phosphorylation of recombinant protein, each of the recombinant proteins (4 µg) was incubated with 4 units of PKAc (purified from bovine heart; Sigma, St. Louis, MO) in a final volume of 20 µl in a kinase buffer containing 25 mM Tris (pH 7.4), 10 mM MgCI₂, 10 µCi, [-³²P]ATP (3,000 Ci/mmol; GE Healthcare, Fairfield, CT) in the presence or absence of PKI (10 µg) (Sigma, St. Louis, MO). A negative control was performed using the same reagents but without the recombinant protein. The reaction mixtures were incubated in 30°C for 30 min, and then 4 µl of 6x SDS sample buffer (60% glycerol, 300 mM Tris [pH 6.8], 12 mM EDTA, 12% SDS, 864 mM 2-mercaptoethanol, and 0.05% bromophenol blue) was added to each tube, which was heated at 100°C for 5 min and then analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. Coomassie blue R250 staining was used to confirm the protein loading equivalency, followed by gel drying and autoradiography.

In silico analysis of PKA phosphorylation site. For candidates interacting with TcPKAc obtained from the yeast two-hybrid screen, the protein sequence was analyzed against PROSITE patterns and profiles (http://us.expasy.org/prosite) to reveal the PKA phosphorylation sites.

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PKA activity is essential for the viability of T. cruzi. Previously, we demonstrated in vitro that TcPKAc was sensitively inhibited by a pseudosubstrate, R-R-X-A, in the peptide of PKI (13). Therefore, pTREX-PKI was introduced into epimastigotes by electroporation. Transfection of this construct into epimastigotes resulted in a lethal effect, while control empty vector (pTREX) or pTREX-GFP transfection resulted in viable parasites. The majority of the parasites transfected with pTREX-PKI died within 4 weeks. On one replicate, a few parasites survived, but examination of these organisms demonstrated that PKI expression was not detectable, indicating these parasites survived by losing PKI expression. We repeated this experiment five times and reproducibly demonstrated that PKI transfection was lethal to the parasites. In addition, we evaluated H89 doses of 5, 10, 15, and 20 µM against epimastigotes. At 10 µM, 98% of the parasites died within 48 h. It takes about 10 days for all of the parasites to be killed. At a dose of 15 µM and 20 µM, it takes from 5 to 7 days to kill all of the parasites. H89 was used as a complementary pharmacological approach to replicate the results seen with PKI transfection.

Identification of TcPKAc interacting proteins. In order to find the downstream interacting proteins or substrates of PKAc in T. cruzi, we performed a large-scale screen using a yeast two-hybrid system and identified 38 candidate interacting proteins (Tables 3, 4, and 5). Table 3 comprises 18 hypothetical proteins with unknown functions. Twelve of these hypothetical proteins contain typical PKA phosphorylation sites. Table 4 consists of 12 genes encoding proteins with putative domains, and six of these proteins contain typical PKA phosphorylation sites. In Table 5, there are eight genes encoding proteins with defined or presumably important functions in this organism. To further characterize these eight genes, the entire ORFs of the genes in Table 5 were subcloned into pAD-Gal4 and retransformed to yeast with pBD-Gal4-TcPKAc. Under the highest stringency selection conditions, all yielded positive colonies. In Fig. 1, these genes are represented in a composite yeast two-hybrid plate.

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TABLE 3. Hypothetical candidate PKA interacting proteins and cAMP-dependent PK phosphorylation site(s)a

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TABLE 4. Candidate PKA interacting proteins with putative domains and cAMP-dependent PK phosphorylation site(s)a

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TABLE 5. Important candidates that interact with TcPKAca

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FIG. 1. Eight candidates with defined or presumably important functions interact with TcPKAc. Using a yeast two-hybrid system, eight genes encoding important proteins in target plasmids interacting with pBD-TcPKAc were obtained. All ORFs of these genes were subcloned into a pAD-Gal4 vector. Each pAD-Gal4 construct was cotransformed into yeast with pBD-TcPKAc and cultured under high-stringency conditions (-Leu, -Trp, -His). These genes are shown on a composite plate, as follows: 1, AQP 9 (TcAQP) (Tc00.1047053508257.140); 2, ATPase (Tc00.1047053508903.100); 3, DERP (Tc00.1047053506983.60); 4, PK (putative ERK homolog) (Tc00.1047053510295.50); 5, hexokinase (Tc00.1047053508951.20); 6, cAMP PDEC2 (GenBank accession no. DQ008164); 7, phosphatidylinositol 3-kinase 2 (PI3 kinase) (Tc00.1047053508859.90); 8, class III phosphatidylinositol 3-phosphate kinase (VPs34) (Tc00.1047053511903.160); 9, positive control (pADwt with pBDwt); and 10, negative control (pADwt with pLaminC).

BLAST analysis of the T. cruzi putative ERK revealed that it had 66% identity with Leishmania major ERK (GenBank accession no. XR_001686644). Since ERK plays an important role in regulating the activities of PDEs to alter the cAMP concentrations in eukaryotic cells, we tested whether these two enzymes interacted. Cotransformation of pAD-Gal4-PDEC2 with pBD-Gal4-ERK was performed in yeast under the highest stringency conditions. Figure 2 demonstrates that these two candidates interacted in this system. With the availability of the TcAQP antibody and the TcPKAc MAb, in Fig. 3, coimmunoprecipitation demonstrated that TcPKAc MAb precipitated a complex containing a 26-kDa band which reacted with the TcAQP antibody, indicating that two proteins, TcPKAc and TcAQP, interacted in this organism.

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FIG. 2. ERK interacted with PDEC2. Cotransformation of pAD-Gal4-PDEC2 with pBD-Gal4-ERK was performed in yeast under the highest stringency conditions. This composite plate demonstrated that these two candidates interacted in this system. 1, pAD-Gal4-PDEC2 with pBD-Gal4-ERK; 2, positive control (pADwt with pBDwt); and 3, negative control (pADwt with pLaminC).

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FIG. 3. TcPKAc MAb interacted with TcAQP in vivo. Coimmunoprecipitation demonstrated that TcPKAc MAb precipitated a complex containing a 26-kDa band which reacted with the TcAQP antibody, indicating that two proteins, TcPKAc and TcAQP, interacted in this organism. 1, negative control using an unrelated MAb (anti-BAG5 of Toxoplasma gondii); 2 and 3, TcPKAc MAb (for details, see Materials and Methods).

PKAc phosphorylates proteins important for T. cruzi growth, adaptation, and differentiation in vitro. The eight genes listed in Table 5 were expressed as N-terminal His₆-tagged recombinant proteins and purified by a HisTrap column precharged with Ni²⁺, and all recombinant proteins were analyzed by SDS-polyacrylamide gel electrophoresis with Coomassie blue R250 staining and verified by immunoblotting with a His₆ MAb (see figure in the supplemental material). In addition, a TcAQP antibody was also used to confirm the expression of the recombinant TcAQP (see figure in the supplemental material). An in vitro phosphorylation analysis demonstrated that PKAc could phosphorylate these eight recombinant proteins and that PKI inhibited the phosphorylation by PKAc in all recombinant proteins (Fig. 4).

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FIG. 4. PKAc phosphorylates important proteins in T. cruzi. In vitro phosphorylation of recombinant proteins important in T cruzi by PKA is confirmed. This composite figure demonstrated that eight proteins were phosphorylated by PKA in vitro. Lane 1, absence of recombinant protein as negative control; lane 2, various recombinant proteins were phosphorylated in the absence of PKI; and lane 3, PKI inhibited the phosphorylation. –, absence of reagent; +, presence of reagent. Arrowheads indicate each of the recombinant proteins and their molecular mass. PI3, phosphatidylinositol 3-kinase 2 (PI3 kinase) (Tc00.1047053508859.90); VPS 34, class III phosphatidylinositol 3-phosphate kinase (VPs34) (Tc00.1047053511903.160); PDEC2, cAMP PDEC2 (GenBank accession no. DQ008164); DERP (Tc00.1047053506983.60); HEXO, hexokinase (Tc00.1047053508951.20); ERK, putative ERK homolog (Tc00.1047053510295.50); ATPase, putative ATPase (Tc00.1047053508903.100); and AQP, AQP 9 (TcAQP) (Tc00.1047053508257.140).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To test whether TcPKAc is essential for the viability of T. cruzi, we transfected a construct expressing PKI which contains R-R-N-A as a PKA-specific pseudosubstrate into epimastigotes. This pseudosubstrate has the same consensus sequence, but instead of a phosphorylatable Ser/Thr residue there is a nonphosphorylatable amino acid, which binds to PKAc and prevents its availability to other substrates. A blockade by this genetic approach killed all parasites repeatedly. Similarly, a pharmacological inhibitor of PKAc, H89, killed epimastigotes at a concentration of 10 µmol. Thus, TcPKAc enzymatic activity is essential for the survival of the parasites.

To understand the biology of TcPKAc, it is important to identify the particular substrates of this enzyme. The yeast two-hybrid system is a useful method to identify protein-protein interactions and has been employed by other investigators to identify PKAc downstream interacting proteins. This approach successfully identified downstream targets of PKAc in the yeast Saccharomyces cerevisiae (22). With the availability of the T. cruzi genome, we can quickly obtain the full sequences of genes identified in our yeast two-hybrid screen and assess the possible physiological functions in this parasite. In this article, we report the identification of 38 TcPKAc interacting genes by this approach. Among these identified candidates, 18 are hypothetical proteins, as their protein sequences do not contain typical functional domains, 12 are putative functional proteins, and 8 are proteins with functions known in mammalian cells and other organisms or T. cruzi. The majority of the identified candidate interacting proteins contain the PKA phosphorylation site (Table 3), and their functions require further investigation. We chose the eight genes with known functions for characterization (Table 5). All eight full-length genes in pAD-Gal4 constructs interacted with pBD-Gal4-TcPKAc in a yeast two-hybrid system under the highest stringency conditions, and PKAc phosphorylated all recombinant proteins from these genes in vitro, indicating that they are substrates of PKAc.

Two PI3 kinase candidates interacted with TcPKAc. PI3 kinase activities have been found in all eukaryotic cells and are connected to an extraordinarily diverse set of key cellular functions, including cell growth, proliferation, motility, differentiation, survival, and intracellular trafficking. A type III PI3 kinase (Vps34) is related with diverse intracellular trafficking events, including autophagy (8). Interestingly, PKA functions have been reported to regulate cell growth, autophagy, and differentiation in the yeast Saccharomyces cerevisiae (3). Phosphorylation of PI3 kinases by PKAc may play a role in regulating PI3 kinase enzymatic activities in T. cruzi.

The regulation of TcAQP, ATPase, and DERP by TcPKAc may play important roles in adaptation to environmental alterations in this parasite. TcAQP has been well characterized for its osmoregulation in T. cruzi. Interestingly, cAMP mediates the translocation of TcAQP to the contractile vacuole complex, resulting in water movement and osmoregulation in T. cruzi (17, 24, 25), and the putative ATPase may also participate in osmoregulation, since T. cruzi encounters immune responses and host free radicals, which will damage its DNA. DNA repair and recombination are important for the viability of this organism, and the regulation of DERP by TcPKAc may serve this purpose.

T. cruzi hexokinase is a typical marker of the glycosome, in which important processes of metabolisms occur (20). By in silico prediction, TcPKAc has a PST1, a peptide sequence at the C terminus that mediates its entry into glycosome (19). T. cruzi hexokinase was characterized and not inhibited by its product, glucose 6-phosphate (4). It is possible that modification of hexokinase by TcPKAc alters its activity and is important for the regulation of this gene. In other organisms, PKA is known to have regulatory effects on metabolic processes, including glucose utilization (28, 31).

Several enzymes regulate cAMP homeostasis in eukaryotic cells (1, 11, 16). For example, during the early developmental events of Dictyostelium spp., ERK is activated by the signals from cell surface receptors. Subsequently, it activates adenylyl cyclase and inhibits RegA (cytoplasmic cAMP PDE), resulting in the elevation of cAMP and the activation of PKAc, and then differentiation proceeds. However, the elevation of PKA activity inhibits ERK activity and enhances PDE activities, thereby forming a regulatory loop. Similar regulations are found in other eukaryotic cells. Here, we demonstrated that PKA interacted with and phosphorylated ERK and PDEC2. Also, ERK and PDEC2 interacted. This suggested that the same genetic network for the regulation of AMP homeostasis might also exist in T. cruzi.

In summary, for the first time, we demonstrated that T. cruzi PKA activity was essential. Moreover, 38 genes which interacted with TcPKAc were identified using a yeast two-hybrid system. Eight of these genes were confirmed to be the substrates of TcPKAc by in vitro phosphorylation. Further studies of PKA substrates should permit us to understand how PKA functions in T. cruzi. The findings in this article open new avenues to study cAMP-PKA pathways in T. cruzi and may eventually lead to novel therapeutic targets for treatment.

 
We thank Alejandra Cecilia Schoijet and Roberto Docampo for providing reagents. Roberto Docampo also kindly provided discussion.

National Institutes of Health grants AI058893, AI076248, and AI05739 supported this work.

 
* Corresponding author. Mailing address: Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2143. Fax: (718) 430-8543. E-mail: huangh{at}aecom.yu.edu

Published ahead of print on 11 August 2008.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: J. F. Urban, Jr.

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Infection and Immunity, October 2008, p. 4757-4763, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00527-08
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