Widefield immunofluorescence microscopy shows retention of exported proteins within the bounds of the PVM. (A) Widefield fluorescence microscopy images of Hyp1-Nluc parasites treated with DMSO (0.1%), Brefeldin A (5μg/ml), Torin 2 (10 nM), MMV396797(4.8μM) or MMV665953 (20.6μM) for 5 h from 20 to 24 hpi. Labelling was performed with mouse anti-EXP2 and rabbit anti-nano luciferase primary antibodies and goat anti-mouse Alexafluor 594 and goat anti-rabbit Alexafluor 488 secondary antibodies. Scale bar=4μm. In trophozoite stage parasites (aged 25–29 hpi) treated with DMSO for5 h, exported Hyp1-Nluc reporter can be seen within the boundary of the host RBC, and what is likely newly synthesized Hyp1-Nluc protein is present within the parasite compartment (A). Following 5-htreatment with Brefeldin A or Torin-2, the Hyp1-Nluc reporter was observed within the bounds of the PVM and appeared to strongly concentrate within the parasite (A). Following treatment with MMV396797, Hyp1-Nluc signal was also concentrated within the para-site(A). (C) Widefield fluorescence images of Hyp1-Nluc parasites treated as specified in (A) from 13 to 15 hpi. Cells were labelled with rabbit anti-EXP2 and mouse anti-GBP130 (WEHI antibody facility) primary antibodies and anti-rabbit Alexafluor 594 and anti-mouse Alexafluor 488 secondary antibodies. Scale bar=4μm. In parasites treated with Brefeldin A, Torin 2 and MMV396797, the majority of the GBP130 signal was retained within the parasite relative to the RBC compartment. In comparison, MMV665953 treatment greatly reduced the anti-GBP130 signal within the parasite in contrast to a modest signal in the RBC space (C).

Looker O, Dans MG, Bullen HE, Sleebs BE, Crabb BS, Gilson PR. The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites. Traffic. 2022 23(9):442-461. PMID: 36040075.