Abstract
Sexual reproduction of Plasmodium parasites takes place in anopheline mosquitoes, where male and female gametes fuse to form zygotes and then ookinetes. These processes are orchestrated by stage-specific protein expression, which is mediated in part by translational repression. Accumulating evidence shows that RNA binding proteins (RBPs) play crucial roles in these processes. Here, we report the characterization of P. berghei 103 (Pb103), which encodes a protein possessing double zinc finger domains (ZFs), an RBP. Reporter parasites expressing azami green fluorescent protein (AGFP) under the endogenous Pb103 gene promoter (Pb103-AGFP reporter) showed that the AGFP fluorescent signal was detected from gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes. The Pb103-disrupted parasites (Pb103(−)) grew and produced gametocytes with similar efficiencies to those of wild-type parasites. However, no oocysts were formed in mosquitoes fed Pb103(−). An in vitro fertilization assay showed abortion at the zygote stage in Pb103(−), suggesting that Pb103 plays a critical role in zygote/ookinete development. Cross-fertilization assays with Pb103(−) and male- or female-sterile parasites revealed that Pb103 was essential exclusively for female gametes. To identify the domains critical for zygote/ookinete development, transgenic parasites expressing partially deleted Pb103 were generated and assayed for ookinete maturation. As a result, deleting either of two ZFs but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression.
Highlights
Malaria is one of the most life-threatening parasitic diseases
We searched PlasmoDB for RNA binding proteins (RBPs)-coding genes whose expression is high in gametocytes to identify novel translation regulators functioning in parasite reproduction stages
A database search showed that P. berghei 103 (Pb103) is highly conserved among rodent malaria parasites (Figure 1A)
Summary
Malaria is one of the most life-threatening parasitic diseases. There are 229 million clinical cases and 409,000 deaths, and the main victims are children under 5 years old living in sub-Saharan Africa [1]. It has been reported that the DDX6class DEAD-box RNA-helicase DOZI and its interacting partner, CITH (CAR-I/Trailer Hitch Homolog) of P. berghei, play a central role in the translational repression of the abovementioned mRNAs at gametocyte stages [18,19]. Deletion mutants of DOZI and CITH can fertilize and differentiate to zygote but fail in ookinete development [19]. DOZI and CITH deletion mutants showed that the expression level of 370 or 183 transcripts, respectively, dropped to less than half because of the instability of these transcripts in the absence of DOZI and CITH [18,19] These demonstrate that the fine tuning of DOZI/CITH-mediated translation is critical for ookinete development. Employing a gene targeting strategy, the function of Pb103 on the parasite life cycle was analyzed with an emphasis on detailed structural characteristics by using partially deleted Pb103 mutants
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