Fig. 2: EXP2 is present in the sporozoite and is translocated to the membrane and secreted after activation.

a Immuno-electron microscopy of sporozoites, stained with rabbit αPfEXP2. Scale bar: 500 nm and 100 nm in insets and transverse views. Representative image of three independent experiments. b Micrographs of permeabilized sporozoites stained with αPfEXP2 (green), αPbTRAP (microneme protein), αPbRON4 (rhoptry protein), or αPbUIS4 (dense granule protein) (all in red) and the DNA dye Hoechst (blue). For EXP2 co-staining with TRAP and UIS4, mouse αPfEXP2 was used. For co-staining with RON4, rabbit αPfEXP2 was used. Scale bar: 5 μm. (>50 sporozoites analyzed per experiment, N = 3 independent experiments). Shown below each panel is the colocalization coefficient between EXP2 and the corresponding protein. c Western blot analysis of secreted EXP2 protein. PbEXP2-HA sporozoites were incubated for 30 min, at 4 °C (−), at 37 °C (+) and in the presence or absence of FCS. Sporozoites were pelleted and both pellet and supernatant were assayed for the presence of PbEXP2-HA, PbCSP (membrane protein of the sporozoite) and PbBiP (ER-resident protein). Lysates of a mixed blood stage infection of PbEXP2-HA were used as control. Representative images of three independent WB. d Number of infected cells at 2 h after infection, infected with WT (white) or EXP2 cKO sporozoites (blue), after treatment with 10 nM rEXP2 concomitantly with sporozoite addition to cells or at 1 h after infection (N = 3 independent experiments totaling nine replicates). e mRNA expression of invasion-related genes of P. berghei at different timepoints during infection, normalized to Pb18S. EXP2 (white), GAP45 (light pink), and EXP1 (dark pink) (N = 2 independent experiments totaling six replicates). f Quantification of EXP2 secretion by sporozoites following activation with FCS at 37 °C throughout time. The amount of EXP2 (blue) in pellet or supernatant fractions for each experiment, was normalized to the amount of CSP in the pellet fraction at the respective time point. Protein extraction was performed immediately after sporozoites were removed from incubation (as in Fig. 2c) and secretion was quantified by WB. Representative images of two independent WB. Results in d and e shown as mean±SEM, two-tailed Mann–Whitney U test was applied for p values.