TGME49_263090

The Toxoplasma gondii 14-3-3 protein (Tg14-3-3) is a parasite-derived effector that drives host cell hypermotility to promote dissemination (PMID:27018989). Tg14-3-3 introduced into cells by parasite-derived fractions, recombinant protein transfection, lentiviral transfer, or regulatable parasite overexpression is each sufficient to induce a hypermotile phenotype in murine and human dendritic cells and microglia, consistent with a 'Trojan horse' mode of spread (PMID:27018989). In tachyzoites, the protein partitions between the cytosol, the membrane fraction, and Triton X-100-resistant lipid raft membranes, with a major cytoplasmic isoform and a minor raft-associated isoform (PMID:12892878). Tg14-3-3 is secreted into the parasitophorous vacuole and released as an excreted/secreted antigen from a pre-existing pool, independent of ongoing protein synthesis (PMID:15109715, PMID:27018989); host 14-3-3 is concurrently recruited to the parasitophorous vacuole membrane in infected dendritic cells (PMID:27018989). The 14-3-3 domain is functionally conserved, complementing a Saccharomyces cerevisiae 14-3-3 mutant (PMID:11182236). Host interactors identified for Tg14-3-3 include the translation initiation factor eIF5B and hCG1821272, linking it to host translation and migration signaling (PMID:29502294).

1. 2001 Medium

   Establishing whether the parasite 14-3-3 homologue is a bona fide functional 14-3-3 rather than a divergent sequence, yeast complementation showed the domain is functionally conserved.

   Evidence Yeast complementation of a S. cerevisiae 14-3-3 mutant with T. gondii cDNA, plus cloning and immunofluorescence

   PMID:11182236

   Open questions at the time

   • Complementation performed in a heterologous yeast system, not in the parasite
   • Native binding partners and phosphopeptide targets not defined
2. 2003 Medium

   To place the protein subcellularly, fractionation revealed cytosolic, membrane, and lipid-raft pools and two isoforms, hinting at compartment-specific roles.

   Evidence Immunofluorescence, subcellular fractionation, and isoform-specific Western blotting of tachyzoites

   PMID:12892878

   Open questions at the time

   • Functional distinction between the two isoforms not resolved
   • Basis of lipid-raft association not defined
3. 2004 Medium

   Whether the protein reaches the host cell was answered by detecting it in the parasitophorous vacuole and excreted/secreted antigens, released from a pre-existing pool.

   Evidence Western blotting of ESA fractions, immunolocalization in infected monocytes, and cycloheximide inhibition assay

   PMID:15109715

   Open questions at the time

   • Secretion pathway and route of release unknown
   • No host target of secreted protein identified at this stage
4. 2016 High

   The central functional question — what Tg14-3-3 does to the host cell — was answered by showing it is sufficient to induce dendritic cell and microglial hypermotility, providing an effector for the Trojan-horse dissemination model.

   Evidence Chromatography/MS identification, recombinant protein transfection, lentiviral transfer, and regulatable overexpression converging on hypermotility; localization in infected DCs

   PMID:27018989

   Open questions at the time

   • Downstream host signaling driving hypermotility not defined
   • Direct host binding partner mediating motility not identified
   • Recruitment of host 14-3-3 to the vacuole membrane mechanistically unexplained
5. 2018 Low

   To identify host machinery targeted by the effector, a yeast two-hybrid screen nominated eIF5B and hCG1821272, implicating host translation and migration pathways.

   Evidence Yeast two-hybrid screen of a human cDNA library with RH-strain Tg14-3-3 bait, with Western blot bait validation

   PMID:29502294

   Open questions at the time

   • Single Y2H screen without confirmatory co-IP or pull-down in mammalian cells
   • Functional consequence of the interactions on host translation or motility not tested
   • Link between these interactors and the hypermotility phenotype not established

• How secreted Tg14-3-3 mechanistically triggers host hypermotility and whether the eIF5B/hCG1821272 interactions execute this remain unresolved.
• No validated host signaling pathway connecting Tg14-3-3 to motility
• Phosphopeptide-binding activity of Tg14-3-3 in host cells untested
• Secretion mechanism and role of the two tachyzoite isoforms unresolved