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CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.

ABSTRACT: Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Here, we show that CD73(-/-) mice, which lack the ability to generate extracellular adenosine, are protected from T. gondii chronic infection, with significantly fewer cysts and reduced susceptibility to reactivation of infection in the CNS independent of host effector function. Parasite dissemination to the brain was unimpaired in CD73(-/-) hosts, suggesting that the reduced cyst number is due to impaired parasite differentiation in the CNS. Confirming this, T. gondii tachyzoites formed fewer cysts following alkaline pH stress in astrocytes isolated from CD73(-/-) mice compared with wild type, and in fibroblasts treated with a CD73 inhibitor. Cyst formation was rescued in CD73(-/-) astrocytes supplemented with adenosine, but not with adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine. Furthermore, mice lacking adenosine receptors had no defect in cyst formation. Based on these findings, we conclude that CD73 expression promotes Toxoplasma bradyzoite differentiation and cyst formation by a mechanism dependent on the generation of adenosine, but independent of adenosine receptor signaling. Overall, these findings suggest that modulators of extracellular adenosine may be used to develop therapies aimed at defending against human toxoplasmosis.

SUBMITTER: Mahamed DA

PROVIDER: S-EPMC3479617 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.

Mahamed Deeqa A DA Mills Jeffrey H JH Egan Charlotte E CE Denkers Eric Y EY Bynoe Margaret S MS

Proceedings of the National Academy of Sciences of the United States of America 20120917 40

Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Her ...[more]

PMID: 22988118

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Project description:UNLABELLED:During infections with the protozoan parasite Toxoplasma gondii, gamma-aminobutyric acid (GABA) is utilized as a carbon source for parasite metabolism and also to facilitate parasite dissemination by stimulating dendritic-cell motility. The best-recognized function for GABA, however, is its role in the nervous system as an inhibitory neurotransmitter that regulates the flow and timing of excitatory neurotransmission. When this pathway is altered, seizures develop. Human toxoplasmosis patients suffer from seizures, suggesting that Toxoplasma interferes with GABA signaling in the brain. Here, we show that while excitatory glutamatergic presynaptic proteins appeared normal, infection with type II ME49 Toxoplasma tissue cysts led to global changes in the distribution of glutamic acid decarboxylase 67 (GAD67), a key enzyme that catalyzes GABA synthesis in the brain. Alterations in GAD67 staining were not due to decreased expression but rather to a change from GAD67 clustering at presynaptic termini to a more diffuse localization throughout the neuropil. Consistent with a loss of GAD67 from the synaptic terminals, Toxoplasma-infected mice develop spontaneous seizures and are more susceptible to drugs that induce seizures by antagonizing GABA receptors. Interestingly, GABAergic protein mislocalization and the response to seizure-inducing drugs were observed in mice infected with type II ME49 but not type III CEP strain parasites, indicating a role for a polymorphic parasite factor(s) in regulating GABAergic synapses. Taken together, these data support a model in which seizures and other neurological complications seen in Toxoplasma-infected individuals are due, at least in part, to changes in GABAergic signaling. IMPORTANCE:Infections of the central nervous system can cause seizures. While inflammation in the brain has been proposed to initiate the onset of the seizures, relatively little is known about how inflammation impacts the structure and function of the neurons. Here we used a parasite called Toxoplasma gondii that infects the brain and showed that seizures arise due to a defect in signaling of GABA, which is the neurotransmitter primarily responsible for preventing the onset of seizures.

Project description:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. Pru?cdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the ?cdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced brain cysts in infected mice, but the numbers of cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored brain cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the brain.

Dataset Information

CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.

Publications

CD73-generated adenosine facilitates Toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system.

Similar Datasets

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