Abstract
After invasion, Toxoplasma resides in a parasitophorous vacuole (PV) that is surrounded by the PV membrane (PVM). Once inside the PV, tachyzoites secrete dense granule proteins (GRAs) of which some, such as GRA16 and GRA24, are transported beyond the PVM likely via a putative translocon. However, once tachyzoites convert into bradyzoites within cysts, it is not known if secreted GRAs can traffic beyond the cyst wall membrane. We used the tetracycline inducible system to drive expression of HA epitope tagged GRA16 and GRA24 after inducing stage conversion and show that these proteins are not secreted beyond the cyst wall membrane. This suggests that secretion of GRA beyond the PVM is not important for the tissue cyst stage of Toxoplasma.
Highlights
Toxoplasma gondii, which belongs to the phylum Apicomplexa, is an obligate intracellular parasite that can cause disease in immuno-compromised patients and fetuses (Montoya and Liesenfeld, 2004; Weiss and Dubey, 2009)
To test if dense granule proteins (GRA) are secreted beyond the tissue cyst wall membrane, we utilized the Tet-inducible system (Etheridge et al, 2014) to express an hemagglutinin epitope tag (HA)-tagged copy of GRA16 or GRA24 under the Tet operator (Tet-O) in parasites expressing Tet-R
We could not just use GRA16-HA or GRA24-HA expressed from the endogenous promoter because if we saw these proteins in the host nucleus we would not know if they were secreted beyond the PV membrane (PVM) before the cyst wall was made or after the cyst wall was made
Summary
Toxoplasma gondii, which belongs to the phylum Apicomplexa, is an obligate intracellular parasite that can cause disease in immuno-compromised patients and fetuses (Montoya and Liesenfeld, 2004; Weiss and Dubey, 2009). Three putative translocon proteins: Myc-regulation 1 (MYR1) along with MYR2 and MYR3 determine transport of GRA16 and GRA24 across the PVM into the host cell cytoplasm after which they traffic to the host cell nucleus (Franco et al, 2016; Marino et al, 2018) In addition to these putative translocon proteins, TgGRA16/24 Don’t Cross Cyst Wall an aspartyl protease, ASP5 cleaves many secreted GRA proteins at a characteristic RRLxx motif known as the Toxoplasma export element (TEXEL) motif which is important for their localization and function (Coffey et al, 2015; Hammoudi et al, 2015). It is unclear if bradyzoites within tissue cysts, akin to tachyzoites within the PV, can secrete GRAs beyond the PVM as the cyst wall is built on the inside of the PVM (Jeffers et al, 2018) and presents a potential barrier for GRA secretion into the host cell
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