<p>(A) mRNA levels of <i>uis2</i> in the <i>P</i>. <i>bergthei</i> life cycle. <i>Pbuis2</i> (<i>PBANKA_132800</i>) mRNA levels were analyzed by real-time PCR using cDNAs from different stages of <i>P</i>. <i>berghei</i>. mRNA level was normalized to arginyl-tRNA synthetase (<i>PbArgRS</i>, PB000094.03.0). MG: midgut; SG: salivary gland; Liver: liver stages; BS: asexual blood stages; Gtc: gametocytes. Each value is the mean ± SD of two independent experiments. (B) Phosphorylation level of eIF2α was higher in <i>uis2</i> cKO Ssp. Levels of UIS2, PbeIF2α-P and total PbeIF2α from 5X10<sup>5</sup> Ssp are shown by immunoblots. CSP was used as a control. Values of densitometry analysis are shown below the bands. This experiment was repeated three times and similar results were obtained. (C) The mutant sporozoites invaded HepG2 cells as effectively as wt parasites. Wild-type TRAP/FlpL(-) and <i>uis2</i> cKO Ssp were added to HepG2 cells and fixed 1 h post-infection. The parasites inside and outside the HepG2 cells were quantified by the hepatocyte invasion assay. Each value is the mean ± SD of two independent experiments. <i>(</i><b><i>D</i></b><i>)</i> The mutant parasites maintained the crescent shape in contrast to the round shape of the wt parasites in HepG2 cells. Wt TRAP/FlpL(-) and <i>uis2</i> cKO Ssp were added to HepG2 cells and were detected 48 h post-infection by immunofluorescence using antibodies against the liver stage antigen UIS4. Bars, 10 μm. (E) The development of <i>uis2</i> cKO sporozoites was blocked inside HepG2 cells. <i>P</i>. <i>berghei</i> Ssp infectivity of HepG2 cells was evaluated 48 h post-infection by counting exo-erythrocytic stage (EEF) numbers and measuring18S rRNA level. The left panel shows the mean of EEF numbers ± SD of two independent experiments. The right panel shows liver-stage parasite burden measured by real-time RT-PCR. (F) The development of <i>uis2</i> cKO sporozoites was blocked in the mouse liver. C57BL/6 mice (five per group) were injected intravenously with 1×10<sup>4</sup> wt TRAP/FlpL(-) or with the same number of <i>uis2</i> cKO Ssp. Liver-stage parasite burden was measured 42 hours post-infection by real-time RT-PCR. Each value is the mean ± SD of two independent experiments.</p
