(A) Alignment of GRA18 alleles from T. gondii strains of types I (TGGT1_288840), II (TGME49_288840), and III (TGVEG_288840). The signal peptide sequence (highlighted in green), the B56 SLiM motifs (LxxLx; boxed in red), and the T. gondii Export Element (TEXEL; RRL motif, boxed in blue) are shown. Single amino acid polymorphisms are indicated by red letters. The alignment was done using ClustalW method. (B) GRA18II-HAFlag in Pru ku80 extracellular parasites is contained in cytoplasmic organelles distinct from the apical micronemes (MIC2) and rhoptries (Toxofilin), and partially co-localizing with the dense granule protein GRA1. (C) GRA18 secretion and export to the host cytoplasm. HFFs were infected with type I RH parasites expressing endogenously tagged GRA18 with hemagglutinin (HA) (upper panel, RHΔku80 GRA18-HA3, in red, Triton X-100 permeabilization) or type II Pru strain ectopically expressing a HAFlag (HF)-tagged copy of GRA18II under the control of the strong promoter of GRA1 (Pru Δku80 PGRA1-GRA18II-HF, ethanol permeabilization). Cells were fixed 18 hr post-infection (hpi) and stained with anti-HA antibodies and Hoechst DNA-specific dye (in blue). The white asterisks indicate uninfected HFF cells. (D) MYR1 is required for GRA18 export in the host cell. HFFs were infected with RH WT or RH Δmyr1 parasites transiently transfected with a vector expressing an HF-tagged GRA18 (PGRA1-GRA18II-HF), and at 18 hpi, the cultures were fixed and stained with antibodies to the HA tag. (E) Schematic representation of GRA18 probability of disorder. Segments with values < 0 are predicted to be disordered (in red), and segments with values > 0 correspond to folded regions (in green).