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Abstract
Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K(m) of 67 μM. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ∼20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ∼20% of its total PSD activity at 37 °C, and the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.
Highlights
Many genes and enzymes of lipid metabolism in T. gondii remain uncharacterized
This study provides evidence for the presence of a novel phosphatidylserine decarboxylase expressed by T. gondii that is soluble and secreted from the parasite into the parasitophorous vacuole (PV) during the intracellular phase of parasite growth
Some soluble forms of phosphatidylserine decarboxylase (PSD) have been reported in bacteria [28], and as a subpopulation of a Plasmodium transgene encoded protein expressed in yeast [10], no secreted forms of the enzyme have been reported previously from any organism
Summary
Many genes and enzymes of lipid metabolism in T. gondii remain uncharacterized. Results: The parasite secretes a soluble phosphatidylserine decarboxylase (TgPSD1), which acts on biological membranes. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ϳ20% of the total PSD, prior to the parasite egress from the host cells. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is found in the parasitophorous vacuole of replicating parasites. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is found in the parasitophorous vacuole of replicating parasites Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism
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