<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts

ABSTRACT 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...

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Autores principales:	Tatsuki Sugi, Vincent Tu, Yanfen Ma, Tadakimi Tomita, Louis M. Weiss
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	amylopectin bradyzoite latency metabolism Toxoplasma gondii energy utilization Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/bb0fb60bac364c72824331c8555dbd54
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spelling	oai:doaj.org-article:bb0fb60bac364c72824331c8555dbd542021-11-15T15:51:44Z<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts10.1128/mBio.01289-172150-7511https://doaj.org/article/bb0fb60bac364c72824331c8555dbd542017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01289-17https://doaj.org/toc/2150-7511ABSTRACT 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. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 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. IMPORTANCE Toxoplasma 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.Tatsuki SugiVincent TuYanfen MaTadakimi TomitaLouis M. WeissAmerican Society for MicrobiologyarticleamylopectinbradyzoitelatencymetabolismToxoplasma gondiienergy utilizationMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	amylopectin bradyzoite latency metabolism Toxoplasma gondii energy utilization Microbiology QR1-502
spellingShingle	amylopectin bradyzoite latency metabolism Toxoplasma gondii energy utilization Microbiology QR1-502 Tatsuki Sugi Vincent Tu Yanfen Ma Tadakimi Tomita Louis M. Weiss <italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
description	ABSTRACT 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. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 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. IMPORTANCE Toxoplasma 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.
format	article
author	Tatsuki Sugi Vincent Tu Yanfen Ma Tadakimi Tomita Louis M. Weiss
author_facet	Tatsuki Sugi Vincent Tu Yanfen Ma Tadakimi Tomita Louis M. Weiss
author_sort	Tatsuki Sugi
title	<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
title_short	<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
title_full	<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
title_fullStr	<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
title_full_unstemmed	<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
title_sort	<italic toggle="yes">toxoplasma gondii</italic> requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of brain cysts
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/bb0fb60bac364c72824331c8555dbd54
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<italic toggle="yes">Toxoplasma gondii</italic> Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts

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