Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

doi:10.1128/mBio.00755-16

. 2016 May 31;7(3):e00755-16.

doi: 10.1128/mBio.00755-16.

Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

Tatsuki Sugi ¹, Yan Fen Ma ², Tadakimi Tomita ², Fumi Murakoshi ³, Michael S Eaton ⁴, Rama Yakubu ², Bing Han ², Vincent Tu ², Kentaro Kato ³, Shin-Ichiro Kawazu ³, Nishith Gupta ⁵, Elena S Suvorova ⁶, Michael W White ⁶, Kami Kim ⁷, Louis M Weiss ⁸

Affiliations

¹ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
² Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA.
³ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
⁴ Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
⁵ Department of Molecular Parasitology, Humboldt University, Berlin, Germany.
⁶ Departments of Molecular Medicine and Global Health, University of South Florida, Tampa, Florida, USA.
⁷ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA.
⁸ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA louis.weiss@einstein.yu.edu.

PMID: 27247232
PMCID: PMC4895117
DOI: 10.1128/mBio.00755-16

Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

Tatsuki Sugi et al. mBio. 2016.

. 2016 May 31;7(3):e00755-16.

doi: 10.1128/mBio.00755-16.

Authors

Tatsuki Sugi ¹, Yan Fen Ma ², Tadakimi Tomita ², Fumi Murakoshi ³, Michael S Eaton ⁴, Rama Yakubu ², Bing Han ², Vincent Tu ², Kentaro Kato ³, Shin-Ichiro Kawazu ³, Nishith Gupta ⁵, Elena S Suvorova ⁶, Michael W White ⁶, Kami Kim ⁷, Louis M Weiss ⁸

Affiliations

¹ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
² Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA.
³ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
⁴ Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.
⁵ Department of Molecular Parasitology, Humboldt University, Berlin, Germany.
⁶ Departments of Molecular Medicine and Global Health, University of South Florida, Tampa, Florida, USA.
⁷ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA.
⁸ Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA louis.weiss@einstein.yu.edu.

PMID: 27247232
PMCID: PMC4895117
DOI: 10.1128/mBio.00755-16

Abstract

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals.

Importance: Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation.

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Figures

FIG 1

FIG 1

TgPKAc3 is distinct from other PKA catalytic subunits in phylogenetic analysis and localization manner. (A) The phylogenetic tree was calculated with the maximum likelihood method based on the Le-Gascuel 2008 model (28). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site (Tg, Toxoplasma gondii; Pf, Plasmodium falciparum; Bb, Babesia bovis; Nc, Neospora caninum; Pm, Perkinsus marinus; Cp, Cryptosporidium parvum; Et, Eimeria tenella; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens). Bootstrap confidence values above 80% are shown in red. (B) RHΔku80Δhxgprt was transfected with C-terminally HA-tagged TgPKAc1, -2, and -3 expression constructs driven by the GRA1 promoter, inoculated into host cells, and incubated for 24 h. Fixed parasites were stained with anti-HA rat MAb 3F10 and anti-IMC1 rabbit antibody followed by detection with Alexa 594-conjugated anti-rat IgG goat secondary antibody or Alexa 488-conjugated anti-rabbit IgG goat secondary antibody. Nuclei were stained with DAPI. Bars, 10 μm.

FIG 2

FIG 2

Localizations of TgPKAcs in extracellular and intracellular parasites. Localizations of TgPKAcs-3HA fusion proteins under the extracellular condition (A) and the bradyzoite condition (B) are shown. (A) TgPKAcs-HA expression plasmids were transfected to RHΔku80Δhxgprt, and the host monolayer was infected with parasites. After a 24-h incubation, parasites were harvested, purified from host cells, and incubated in the culture medium for 30 min at 37°C on the glass coverslips. Attached parasites were fixed and stained with anti-HA and IMC marker GAP45. (B) PruΔku80Δhxgprt parasites were transfected with TgPKAc-HA expression plasmids and inoculated into the host monolayer. After a 2-h invasion, infected host cells were washed and cultured in pH 8.2 medium under a CO₂-depletion condition for 48 h. Fixed infected host cells were stained with anti-HA antibody and anti-GFP antibody. Bars, 10 μm. DIC, differential interference contrast.

FIG 3

FIG 3

In vitro kinase assay of PKAc1 and PKAc3 kinase domain. pcDNA3 mammalian expression plasmid containing coding sequence of HA-tagged PKAc1 and a kinase domain of PKAc3 (PKAc3-Δ120) was transfected to 293T cells, incubated for 48 h, and lysed to purify the recombinant proteins by anti-HA tag immunoprecipitation. The immunoprecipitated TgPKAc1-HA or TgPKAc3-Δ120-HA complex was used for an in vitro kinase assay in the presence of 1.66 μM cAMP, 1 μM PKA inhibitor peptide PKI, PKC-CAMK inhibitor mixture (3.3 μM PKC inhibitor peptide and 0.33 μM R24571), or 10 μM H89 or in the absence of cAMP according to the manufacturer’s protocol for the PKA assay kit (Merck Millipore). Incorporation of radioactive ³²P into the phosphorylated PKA substrate peptide Kemptide was measured with a scintillation counter. Relative PKA kinase activity was normalized with the average value from the PKAc1-HA reaction in the presence of cAMP without any inhibitors. As a control, mock-transfected 293T cell lysate was used for immunoprecipitation, and the kinase assay was performed (rightmost bar). Average values from a representative of two independent experiments are shown.

FIG 4

FIG 4

Knockout of TgPKAc3 and its complementation. (A) Schematic depiction of the manipulated TgPKAc3 locus. The knockout construct contains HXGPRT-expressing cassette bound with 5′ untranslated region and 3′ untranslated region of TgPKAc3 (green filled and open boxes, respectively). The genomic locus of TgPKAc3 was replaced with HXGPRT by double homologous recombination and produced PKAc3KO. The complementation construct contains 2.2-kbp 5′ untranslated region and genomic DNA sequence spanning TgPKAc3 coding sequence (CDS) just before the stop codon followed by the 3HA tag (red box) and terminator from HXGPRT (blue open box). The drug selectable marker is shown in the orange open box. The NotI site just after the DHFR selectable marker is shown. Detection primers to check the transgenic parasite clones are shown by arrows. (B) Genomic DNA from parental parasite PruΔku80Δhxgprt (Parent), knockout clone PruΔku80Δpkac3 (PKAc3KO), and complemented clone PruΔku80Δpkac3::PKAc3-3HA (COMP) was used for PCR amplification using the primer set described at bottom. (C) Forty-eight hours after parasite inoculation, infected host cells were stained with anti-HA antibody (red). GFP signals driven by LDH2 were enhanced with staining with anti-GFP antibody (green), and nuclei were stained with DAPI (blue). Bars, 10 μm. (D) 3HA-tagged TgPKAc3 was detected from the protein lysate from parental parasite PruΔku80Δhxgprt (Parent), knockout clone PruΔku80Δpkac3 (PKAc3KO), and complemented clone PruΔku80Δpkac3::PKAc3-3HA (COMP). Protein lysate from 10⁶ parasites/lane was loaded and detected with anti-HA (left panel), anti-GRA1 (center panel), and anti-GFP (right panel) antibodies. Arrows show the four distinct bands detected in the TgPKAc3-3HA complemented parasite. WT, wild type; KO, knockout; MW, molecular weight in thousands.

FIG 5

FIG 5

Growth of the TgPKAc3 knockout under normal culture conditions. (A) Overall growth speed was measured by plaque assay. Forty parasites for PruΔku80Δhxgprt and PruΔku80Δpkac3::PKAc3-3HA or 80 parasites for PruΔku80Δpkac3 were inoculated into HFF and incubated for 14 days. (B) Parasites were allowed to invade the host cells for 30 min and fixed, and extracellular parasites and total parasites were stained sequentially. Invaded parasites/total parasites are shown as invasion rate. (C and D) Parasite numbers within vacuoles were counted at 10 h (C) or 18 h (D) after inoculation. Ratios of parasite number to vacuole are shown. If one-way analysis of variance detected a significant difference within parent, Δpkac3, and complemented strain values, then Tukey’s honestly significant difference test results are shown. *, P < 0.05; **, P < 0.01.

FIG 6

FIG 6

Disruption of TgPKAc3 induces bradyzoite-specific cyst wall formation. The cyst wall was stained to measure the bradyzoite differentiation status in the parental parasite PruΔku80Δhxgprt (Parent), knockout clone PruΔku80Δpkac3 (PKAc3KO), and complemented clone PruΔku80Δpkac3::PKAc3-3HA (COMP). Infected host cells were grown under normal culture conditions or bradyzoite induction conditions (pH 8.2 and CO₂ depletion) for 48 h. Parasites were stained with anti-CST1 antibody (shown in green) or anti-Toxoplasma serum (shown in red). (A) Representative images of vacuoles with cyst walls (center panel, PruΔku80Δpkac3) and without cyst walls (left and right panels, showing PruΔku80Δhxgprt and PruΔku80Δpkac3::PKAc3-3HA, respectively) under tachyzoite culture condition. (B) Cyst wall-positive vacuoles were identified as parasitophorous vacuoles that have anti-CST1 signal associated with the parasitophorous vacuole. Quantitative measurements of the cyst wall-positive vacuole rate are shown. At least 100 total vacuoles per sample were counted, and cyst wall-positive vacuoles per total vacuoles are shown. Mean values and standard deviations from independent triplicate experiments are shown. If one-way analysis of variance detected significant differences among the group, Tukey’s honestly significant difference test results are shown. *, P < 0.05; **, P < 0.01; n.s., nonsignificant.

FIG 7

FIG 7

TgPKAc3 affects bradyzoite differentiation in the RH strain. The cyst wall was stained to measure the bradyzoite differentiation status in the parental parasite RHΔku80Δhxgprt (Parent) and knockout clone RHΔku80Δpkac3 (RH Δpkac3). Infected host cells were grown under normal culture conditions (A) or bradyzoite induction conditions (pH 8.2 and complete CO₂ depletion) (B) for 48 h. Parasites were stained with anti-CST1 antibody and anti-Toxoplasma serum. Cyst wall-positive vacuoles were identified as parasitophorous vacuoles that have anti-CST1 signal associated with the parasitophorous vacuole. Quantitative measurements of the cyst wall-positive vacuole rate are shown. At least 100 total vacuoles per sample were counted, and cyst wall-positive vacuoles per total vacuoles are shown. Mean values and standard deviations from independent triplicate experiments are shown. Statistical differences determined by Student’s t test between parent and TgPKAc3KO are shown (**, P < 0.01).

FIG 8

FIG 8

Long-term elevation of cAMP causes tachyzoite maintenance via TgPKAc3 signal. After a 2-h invasion window, parasites were treated with IBMX (500 μM final concentration) or vehicle control DMSO for 48 h. The cyst wall was stained to measure the bradyzoite differentiation status in the parental parasite PruΔku80Δhxgprt (Parent), knockout clone PruΔku80Δpkac3 (PKAc3KO), and complemented clone PruΔku80Δpkac3::PKAc3-3HA (COMP). Cyst wall-positive vacuoles were identified as parasitophorous vacuoles that have anti-CST1 signal associated with the parasitophorous vacuole as shown in Fig. 5A. At least 100 total vacuoles per sample were counted, and percentages of cyst wall-positive vacuoles per total vacuoles are shown. Mean values and standard deviations from independent triplicate experiments are shown. Student’s t test between DMSO and IBMX treatment was performed. *, P < 0.05; **, P < 0.01.

FIG 9

FIG 9

In vivo cyst production with TgPKAc3 mutant parasites. Six- to 8-week-old female C57BL/6J mice were infected with 10,000 parasites via the intraperitoneal route. Seven weeks after infection, the tissue cyst number in the infected mouse brain was counted. The average numbers of cysts and standard deviations (n = 4 for wild type [WT], n = 3 for knockout [KO], and n = 4 for complemented) are shown. Mean values were statistically analyzed with one-way analysis of variance for detecting a difference in the groups and with a post hoc Tukey honestly significant difference test for pairwise comparison. n.s., P > 0.05; *, P < 0.05; **, P < 0.01.

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References

1. Pappas G, Roussos N, Falagas ME. 2009. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol 39:1385–1394. doi:10.1016/j.ijpara.200904003. - DOI - PubMed
1. Dubey JP, Frenkel JK. 1976. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J Protozool 23:537–546. doi:10.1111/j.1550-7408.1976.tb03836.x. - DOI - PubMed
1. Sakikawa M, Noda S, Hanaoka M, Nakayama H, Hojo S, Kakinoki S, Nakata M, Yasuda T, Ikenoue T, Kojima T. 2012. Anti-Toxoplasma antibody prevalence, primary infection rate, and risk factors in a study of toxoplasmosis in 4,466 pregnant women in Japan. Clin Vaccine Immunol 19:365–367. doi:10.1128/CVI.05486-11. - DOI - PMC - PubMed
1. Weiss L, Kim K. 2014. Toxoplasma gondii. The model apicomplexan—perspectives and methods, 2nd ed. Academic Press (Elsevier Inc.), London, United Kingdom.
1. Ferreira da Silva MDF, Barbosa HS, Gross U, Lüder CGK. 2008. Stress-related and spontaneous stage differentiation of Toxoplasma gondii. Mol Biosyst 4:824–834. doi:10.1039/b800520f. - DOI - PubMed

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[1] Pappas G, Roussos N, Falagas ME. 2009. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol 39:1385–1394. doi:10.1016/j.ijpara.200904003. - DOI - PubMed

[2] Pappas G, Roussos N, Falagas ME. 2009. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol 39:1385–1394. doi:10.1016/j.ijpara.200904003. - DOI - PubMed

[3] Dubey JP, Frenkel JK. 1976. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J Protozool 23:537–546. doi:10.1111/j.1550-7408.1976.tb03836.x. - DOI - PubMed

[4] Dubey JP, Frenkel JK. 1976. Feline toxoplasmosis from acutely infected mice and the development of Toxoplasma cysts. J Protozool 23:537–546. doi:10.1111/j.1550-7408.1976.tb03836.x. - DOI - PubMed

[5] Sakikawa M, Noda S, Hanaoka M, Nakayama H, Hojo S, Kakinoki S, Nakata M, Yasuda T, Ikenoue T, Kojima T. 2012. Anti-Toxoplasma antibody prevalence, primary infection rate, and risk factors in a study of toxoplasmosis in 4,466 pregnant women in Japan. Clin Vaccine Immunol 19:365–367. doi:10.1128/CVI.05486-11. - DOI - PMC - PubMed

[6] Sakikawa M, Noda S, Hanaoka M, Nakayama H, Hojo S, Kakinoki S, Nakata M, Yasuda T, Ikenoue T, Kojima T. 2012. Anti-Toxoplasma antibody prevalence, primary infection rate, and risk factors in a study of toxoplasmosis in 4,466 pregnant women in Japan. Clin Vaccine Immunol 19:365–367. doi:10.1128/CVI.05486-11. - DOI - PMC - PubMed

[7] Weiss L, Kim K. 2014. Toxoplasma gondii. The model apicomplexan—perspectives and methods, 2nd ed. Academic Press (Elsevier Inc.), London, United Kingdom.

[8] Weiss L, Kim K. 2014. Toxoplasma gondii. The model apicomplexan—perspectives and methods, 2nd ed. Academic Press (Elsevier Inc.), London, United Kingdom.

[9] Ferreira da Silva MDF, Barbosa HS, Gross U, Lüder CGK. 2008. Stress-related and spontaneous stage differentiation of Toxoplasma gondii. Mol Biosyst 4:824–834. doi:10.1039/b800520f. - DOI - PubMed

[10] Ferreira da Silva MDF, Barbosa HS, Gross U, Lüder CGK. 2008. Stress-related and spontaneous stage differentiation of Toxoplasma gondii. Mol Biosyst 4:824–834. doi:10.1039/b800520f. - DOI - PubMed

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Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

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Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

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