Phosphorylation of a Toxoplasma gondii tyrosine transporter by calcium-dependent kinase 3 is important for parasite fitness

Abstract

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. The calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout ({Delta}cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with {Delta}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, a putative transporter of the major facilitator superfamily (MFS) and show that it is rapidly phosphorylated after induction of calcium signalling. Conditional knockout (KO) of the transporter reveals an essential role in the lytic cycle during intracellular growth with a transcriptome signature of amino acid-starved parasites. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import. Complementation with phosphorylation site mutants shows that phosphorylation of serine 56 (S56) by TgCDPK3 gives the parasites a growth benefit in competition assays. Collectively, these findings validate an important, albeit non-essential role for TgCDPK3 in the regulation of metabolic processes, in addition to motility.\n\nAuthor summaryToxoplasma gondii is an obligate intracellular parasite. To survive and spread throughout the host it must repeatedly infect, replicate within and exit, host cells. These recurring cycles of infection and egress rely on signalling pathways that allow the parasites to sense and respond rapidly to their environment. While some key kinases and secondary messengers within these pathways have been identified, functional analysis of non-kinases has been very limited. This is especially true for candidates that are not predicted to play a role in active motility or are not known to function in established signalling pathways. Here we have followed up on an unexpected target of the T. gondii calcium-dependent kinase 3 (TgCDPK3), a plant-like calcium dependent kinase, that was previously shown to play an important role in calcium-mediated exit from the host cell. We show that, in addition to controlling motility of the parasite (as previously shown), TgCDPK3 phosphorylates an essential tyrosine transporter in the plasma membrane. Mutational analysis of the phosphorylation sites demonstrates an important role in maintaining parasite fitness, thus demonstrating that TgCDPK3 plays a pleiotropic role in controlling both egress and metabolism.