Abstract
Summary Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium‐dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3‐dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5‐3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5‐3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non‐phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5‐3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.
Highlights
The fast-growing tachyzoite stage of the protozoan parasite Toxoplasma gondii requires cycles of host cell invasion, replication and lysis for its successful proliferation within the host
ApiAT5-3 is located at the parasite periphery and phosphorylated during ionophore-induced egress in a TgCDPK3-dependent manner
ApiAT5-3 was previously identified as phosphorylated at serine 56 in a TgCDPK3-dependent manner (Treeck et al, 2014)
Summary
The fast-growing tachyzoite stage of the protozoan parasite Toxoplasma gondii requires cycles of host cell invasion, replication and lysis for its successful proliferation within the host Each step of this lytic cycle involves tightly regulated signalling pathways, the intricacies of which remain largely unknown. Ca2+ flux regulates key processes including secretion of micronemes prior to host cell entry (Carruthers and Sibley, 1999), parasite motility (Wetzel et al, 2004), and host cell egress (Endo et al, 1982) and invasion (Lovett and Sibley, 2003) These processes can all be inhibited by Ca2+ immobilisers or chelators, such as BAPTA-AM (Mondragon and Frixione, 1996; Black et al, 2000; Moudy et al, 2001; Wetzel et al, 2004). It is anchored to the parasite plasma membrane, via N-terminal myristoylation and palmitoylation motifs
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