Abstract
ABSTRACTIn 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.
Highlights
In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis
We introduced point mutations at a phosphorylation site in TgGP that we predicted would be involved in the regulation of TgGP enzymatic activity to create mutants with inactive or hyperactive glycogen phosphorylase (GP), tested these mutant parasites in an in vivo mouse model infection to evaluate the effects of manipulation of TgGP on amylopectin digestion/storage, and elucidated the effects of amylopectin abnormalities on the ability of this parasite to develop a latent infection in the murine brain
To investigate the role of amylopectin regulation in this parasite, we focused on TgGP, which is a digestive enzyme for starch (Fig. 1A) [9]
Summary
Latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis. 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. 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. Immunocompetent humans can eventually suppress tachyzoite replication, conversion of this parasite to bradyzoites (within tissue cysts) results in chronic infection, and these latent organisms evade control by the host immune system [1]. It is not clear how T. gondii regulates the enzymes involved in amylopectin metabolism to establish a regulated system of amylopectin storage and digestion
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