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. 2020 Oct 20;11(5):e02092-20. doi: 10.1128/mBio.02092-20

FIG 5.

FIG 5

TgVps45 is essential for ingestion and digestion of cytosolic host proteins. (A) IFA of TgVps45-mAID-HA parasites ± IAA (24 hours). ELC homeostasis is grossly affected in the absence of TgVps45. proM2AP, ELC; actin, parasite cytosol. (B) IAA-induced TgVsp45-mAID-HA degradation in extracellular parasites within 2 hours. Catalase, loading control. (C) Parasites lacking TgVps45 are not impaired in invasion. Error bars represent the ± SD for three independent experiments. (D) Quantification of ingestion of host cytosolic GFP 7 minutes postinfection in WT or Vps45-mAID-HA knockdown ± IAA parasites. The percentages of GFP-positive tachyzoites from three independent experiments are shown. (E) IFA of Vps45-mAID-HA parasites + IAA (30 minutes postinfection of GFP-positive host cells). Colocalization of ingested GFP with the VAC was rarely observed. CPL, VAC; actin, parasite cytosol. (F) Quantification of ingestion of host cytosolic GFP 30 minutes postinfection in Vps45-mAID-HA knockdown ± IAA parasites ± LHVS. The percentages of GFP-positive tachyzoites from three independent experiments are shown. (G to H) The VAC morphology is dynamic and can appear as a single dot or a scattered pattern. The morphology of VAC is affected following depletion of TgVps45 (G). Error bars represent the ± SD for three independent experiments. Representative images are shown in panel H. CPL, VAC; GAP50, parasite IMC. (I) Electron microscopy reveals an enlarged micropore in Vps45 conditional knockdown (cKD) parasites. Arrowhead, micropore. Scale bars = 1 μm. Scale bars for all IFA = 7 μm.