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Differential protein phosphorylation during stage conversion in Toxoplasma gondii

ABSTRACT: The intracellular parasite Toxoplasma gondii resides within a membrane bound parasitophorous vacuole and secretes an array of proteins to establish this replicative niche during acute stage of infection. When rapidly dividing tachyzoites convert to chronic stage bradyzoites, many secreted proteins are reported to dynamically redistribute as the cyst forms and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analysis comparing tachyzoite and bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. These data support a model in which secreted kinases and phosphatases are important to dynamically regulate parasite secreted proteins during stage conversion.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Toxoplasma Gondii Me49 Homo Sapiens (human)

TISSUE(S): Foreskin Fibroblast, Cell Culture

DISEASE(S): Toxoplasmosis

SUBMITTER: Malgorzata Broncel

LAB HEAD: Moritz Treeck

PROVIDER: PXD019729 | Pride | 2020-06-12

REPOSITORIES: Pride

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Project description:Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling of FV-P6 mutant parasites at 35C and 40C, as well as FV-P6 complemented with a wild-type Cactin encoding cosmid (TOXO93 or comp) and as a pseudo-diploid wild-type line encoding both the wild-type and mutant Cactin locus (S4.2), also at both temperatures. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. Since TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development. The role of Cactin in G1 progression of Toxoplasma gondii tachyzoites was assessed by expression profiling a temperature senstive mutant of Cactin at both the permissive (35C) as well as the restrictive (40C) temperature. Controls under the same temperature conditions inlcude the mutant line complemented with a wild-type Cactin allele encoded on a cosmid and a wild-type parasite line expressing an additional, mutant allele of Cactin (pseudo-diploid line).

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Differential protein phosphorylation during stage conversion in Toxoplasma gondii

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