Stage-specific transcription of the PbeIF2α kinases. PbeIF2α kinases (PbeIK1, PbeIK2, and PbPK4) transcript levels were analyzed by real-time PCR using cDNAs from different stages of P. berghei. Each value is the mean ± SD of two independent experiments.

Stress granules in salivary gland sporozoites are PbeIK2 dependent. (A) The majority of wild-type salivary gland sporozoites contain poly(A) RNA granules. GFP-expressing sporozoites were subjected to oligo-d(T) in situ hybridization followed by fluorescence microscopy. (left) A higher magnification image. (B) Stress granule–associated proteins eIF2α-P and PABP were revealed by immunofluorescence in wild-type salivary gland sporozoites, but were absent in PbeIK2 (-) sporozoites. (left) A higher magnification image. In situ hybridization and immunofluorescence assays were repeated in at least three independent experiments.

PbeIK2 leads to translational repression in salivary gland sporozoites. (A) 5 × 10⁵ sporozoites of wild-type P. berghei were dissected from mosquito midguts, hemocoels, and salivary glands. Levels of PbeIF2α-P and total PbeIF2α were revealed by densitometry analysis of immunoblots. Values are shown below the bands. (B) PbeIF2α phosphorylation levels of wild-type and PbeIK2 (-) salivary gland sporozoites were assayed by immunoblots. (C) The same number (5 × 10⁵) of wild-type and PbeIK2 (-) salivary gland sporozoites were exposed to ³⁵S-Met/Cys at 37°C for 30 min. Samples were then resolved on SDS-PAGE for autoradiography. (D) Effect of temperature on the kinetic incorporation of ³⁵S- Met/Cys by wild-type and PbeIK2 (-) salivary gland sporozoites. These data were obtained in two independent experiments.

Enhanced liver stage proteins expression in PbeIK2 (-) salivary gland sporozoites. (A) Wild-type and PbeIK2 (-) sporozoites were incubated at 37°C for different times, then UIS4, HSP70, and CSP were visualized on immunoblots by using the polyclonal UIS4 antisera, mAb 2E6, and 3D11, respectively. Antiserum against total PbeIF2α served as loading controls. (B) Sporozoites were incubated at 25°C for different times, and the amounts of CSP secreted into the culture medium were immunoblotted with mAb 3D11. (C) CSP in wild-type and PbeIK2 (-) midgut sporozoites. On day 14 after the first infectious blood meal, wild-type and PbeIK2 (-) parasites were dissected from mosquito midguts, and CSP was visualized on immunoblot. Immunoblots were repeated at least three independent experiments.

Morphology of PbeIK2 (-) salivary gland sporozoites. (A) Fluorescence view of wild-type and PbeIK2 (-) sporozoites fixed immediately after dissection from mosquito salivary glands or after 4 h incubation at 37°C. Bars, 10 µm. (B) Sal treated wild-type sporozoites were incubated at 37°C for different hours. Numbers of transformation bulbs were counted. One group of sporozoites was incubated with 50 µM proteasome inhibitor MG132 for 4 h. These data were obtained from two independent experiments. (C) TEM of PbeIK2 (-) sporozoites fixed immediately after dissection from mosquito salivary glands. Mn, Micronemes. Panels i and ii, wild-type; iii-vi, mutant. Arrows in panels iii and iv point to bulbs; in panel v, membrane detachment; in panel vi, transformation bulb with redistribution of organelles. Bars, 1 µm.

Phenotype of PbeIK2 (-) sporozoites. (A) PbeIK2 (-) parasites develop normally in asexual blood stages. Swiss Webster mice (5 per group) were injected intraperitoneally with 200 µl of blood infected with PbeIK2 (-) or wild-type parasites (1% parasitemia). The parasitemia of the recipient mice was checked in Giemsa-stained blood smears. (B) PbeIK2 (-) and wild-type parasites produce the same number of midgut and salivary gland–associated sporozoites on day 14 and 20 after the first infectious blood meal, respectively. Sporozoite numbers were counted in three different mosquito cycles of the wild type and mutants. (C) PbeIK2 (-), and wild-type–infected mosquitoes release saliva containing the same number of sporozoites into the medium. Sporozoite numbers were counted in three independent experiments. (D) C57BL/6 mice (five mice per group) were intravenously injected with 1 × 10⁴ wild type or either one of two clones of PbeIK2 (-) sporozoites (ko1 and ko2). Liver-stage parasite burden was measured by real-time RT-PCR, and shown are the means ± SD of two independent experiments. (E) In three independent experiments 2 × 10⁴ or 2 × 10³ wild-type and PbeIK2 (-) sporozoites were added to HepG2 cells. Infectivity of sporozoites to HepG2 cells were evaluated by counting EEF numbers 48 h after infection.

Evidence for the presence of a PbeIF2α phosphatase in salivary gland sporozoites. (A) Wild-type sporozoites were incubated at 37°C for 30 and 60 min, and then levels of PbeIF2α-P and total PbeIF2α were assayed by immunoblots. In B–E, wild-type and PbeIK2 (-) sporozoites were dissected from mosquito salivary glands in the presence (+) or absence (-) of 1 uM salubrinal (Sal) at room temperature, and then the medium was replaced by DME supplemented with 10% FBS. (B) Immunoblot assays for levels of PbeIF2α-P and total PbeIF2α of wild-type and PbeIK2 (-) sporozoites treated with or without Sal. In A and B, immunoblots were repeated at least four times and densitometry values are shown below bands. (C) Infectivity of Sal-treated sporozoites to HepG2 cells were evaluated by counting EEF numbers 48 h after infection. Data are shown as the means ± SD of three independent experiments. (D) Sal enhanced infectivity of wild-type sporozoites to mice. C57BL/6 mice (six mice per group) were intravenously injected with 5 × 10³ wild-type sporozoites treated with or without Sal. 42 h later, liver parasite burden was measured by real-time PCR. Data are shown as the means ± SD of two independent experiments. (E) Effect of Sal on the kinetic incorporation of ³⁵S-Met/Cys by wild-type and PbeIK2 (-) salivary gland sporozoites at 37°C. Data are shown as the means ± SD of two independent experiments.