Utilization of inherent miRNAs in functional analyses of Toxoplasma gondii genes

MicroRNAs (miRNAs) are crucial genetic effectors partaking in numerous mechanisms of gene regulation in eukaryotic organisms. Recent discoveries of miRNA in Toxoplasma gondii, an intracellular obligate parasite of the phylum Apicomplexa, suggested possible roles of T. gondii miRNAs (Tg-miRNAs) in the post-transcriptional gene regulation and in the cell biology of the parasite. To gain a better understanding of the involvement of Tg-miRNAs in regulating the parasite gene expression, a dual luciferase reporter system was used in the examination and evaluation of the effects of endogenous Tg-miRNAs, their mimics and inhibitors. A Renilla luciferase (Rnluc) transcript was engineered to carry independent binding sites of two abundant species, namely Tg-miR-60a and Tg-miR-4a, so that the expression of Rnluc was silenced in a sequence specific manner byTg-miR-60a and Tg-miR-4a. Notably, Tg-miR-60a, but not Tg-miR-4a, caused the levels of Rnluc transcripts to decrease. These findings strongly suggested that T. gondiiemploys the Tg-miRNA species-specific mode of silencing actions: transcript degradation by Tg-miR-60a, and translational suppression by Tg-miR-4a. Herein we developed a genetic system that exploits and directs the most abundant Tg-miR-60a for loss-of-function analyses in T. gondii. As a proof of principle, we showed that when the binding sites for Tg-miR-60a were introduced into the parasite transcripts via homologous recombination at the locus of (i) DEAD-box RNA helicase (TgHoDI), or (ii) lactate dehydrogenase isoform 1 (TgLDH1), the expression levels of the selected genes can be altered. It was thus proven that inherit Tg-miR-60a could be directed and used to assist in the loss-of-function analyses

Complex nutrient channel phenotypes despite Mendelian inheritance in a Plasmodium falciparum genetic cross

Malaria parasites activate a broad-selectivity ion channel on their host erythrocyte membrane to obtain essential nutrients from the bloodstream. This conserved channel, known as the plasmodial surface anion channel (PSAC), has been linked to parasite clag3 genes in P. falciparum, but epigenetic switching between the two copies of this gene hinders clear understanding of how the encoded protein determines PSAC activity. Here, we used linkage analysis in a P. falciparum cross where one parent carries a single clag3 gene to overcome the effects of switching and confirm a primary role of the clag3 product with high confidence. Despite Mendelian inheritance, CLAG3 conditional knockdown revealed remarkably preserved nutrient and solute uptake. Even more surprisingly, transport remained sensitive to a CLAG3 isoform-specific inhibitor despite quantitative knockdown, indicating that low doses of the CLAG3 transgene are sufficient to confer block. We then produced a complete CLAG3 knockout line and found it exhibits an incomplete loss of transport activity, in contrast to rhoph2 and rhoph3, two PSAC-associated genes that cannot be disrupted because nutrient uptake is abolished in their absence. Although the CLAG3 knockout did not incur a fitness cost under standard nutrient-rich culture conditions, this parasite could not be propagated in a modified medium that more closely resembles human plasma. These studies implicate oligomerization of CLAG paralogs encoded by various chromosomes in channel formation. They also reveal that CLAG3 is dispensable under standard in vitro conditions but required for propagation under physiological conditions