Abstract
BackgroundToxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. Understanding the mechanism of tachyzoite invasion is important for the discovery of new drug targets and may serve as a model for the study of other apicomplexan parasites. We previously showed that Plasmodium falciparum expresses a homolog of human calcium calmodulin-dependent protein kinase (CaMK) that is important for host cell invasion. In this study, to identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite.MethodsAn in vitro kinase assay was performed to assess the phosphorylation activities of a novel CaMK-like protein in T. gondii by using purified proteins with various concentrations of calcium, calmodulin antagonists, or T. gondii glideosome proteins. Indirect immunofluorescence microscopy was performed to detect the localization of this protein kinase by using the antibodies against this protein and organellar maker proteins of T. gondii.ResultsWe identified a novel CaMK homolog in T. gondii, T. gondii CaMK-related kinase (TgCaMKrk), which exhibits calmodulin-independent autophosphorylation and substrate phosphorylation activity. However, calmodulin antagonists had no effect on its kinase activity. In T. gondii-infected cells, TgCaMKrk localized to the apical ends of extracellular and intracellular tachyzoites. TgCaMKrk phosphorylated TgGAP45 for phosphorylation in vitro.ConclusionsOur data improve our understanding of T. gondii motility and infection, the interaction between parasite protein kinases and glideosomes, and drug targets for protozoan diseases.
Highlights
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis
We identified a homolog of P. falciparum protein kinase 2 (PfPK2) in T. gondii, T. gondii calmodulin-dependent protein kinase (CaMK)-related kinase (TgCaMKrk) (ToxoDB ID: TGME49_315190; GenBank accession number: AB699221), which exhibits autophosphorylation and histone phosphorylation activity
We further show that TgCaMKrk is expressed in T. gondii-infected cells and localizes to the apical ends of extracellular and intracellular tachyzoites, and that it targets TgGAP45 for phosphorylation in vitro
Summary
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa and a major pathogen of animals and immunocompromised humans, in whom it causes encephalitis. To identify novel targets for the treatment of Toxoplasma gondii infection (another apicomplexan parasite), we sought to identify a CaMK-like protein in the T. gondii genome and to characterize its role in the life-cycle of this parasite. Additional T. gondii protein kinases are involved in host manipulation, cell cycle regulation, and functions required for growth, stress responses, and the transition from tachyzoite to bradyzoite [15]. Given their level of involvement in many aspects of the parasitic life-cycle, the kinases encoded by the parasite genome are obvious potential drug targets
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