The RNAi-Competent Malaria Parasite: A Novel Strategy to Knock Down Plasmodium Genes via Non-Canonical RNAi

Abstract

Malaria, caused by apicomplexan parasites of the Plasmodium species, is one of the deadliest infectious diseases worldwide. Despite the urgent need to identify new drug targets and vaccine candidates, a large proportion of the Plasmodium genes are uncharacterized, as tools to study gene function are limited. In many eukaryotes, genes can be silenced via RNA interference (RNAi) using artificial short hairpin RNAs (shRNAs). However, Plasmodium parasites lack the machinery required for RNAi. In this study, I therefore engineered a non-canonical RNAi machinery into the rodent parasite Plasmodium berghei (P. berghei). To this end, I exploited a non-canonical RNAi pathway which requires only a single protein, Argonaute 2 (Ago2), and a specifically designed shRNA, a so-called AgoshRNA, for gene silencing. I generated a P. berghei line constitutively expressing Ago2, named PbAgo2, and demonstrated that this parasite can complete its life cycle through the mammalian and insect host, despite exhibiting a reduced growth in blood and mosquito stages. Expression of AgoshRNAs targeting the mRNA of the green fluorescent protein GFP (constitutively expressed by PbAgo2) induced a potent knockdown of GFP both in blood and in non-erythrocytic stages. As different AgoshRNAs mediated gene silencing to various levels, target gene expression could be fine-tuned. AgoshRNA-mediated gene knockdown was also possible for endogenous genes, and the knockdown of a non-essential gene phenocopied the full knockout. Additionally, the expression of a blood-stage-essential gene was reduced using RNAi. The analysis of the transcriptome of PbAgo2 by RNA sequencing suggested a possible interaction between Ago2 and a Plasmodium mRNA storage protein as a putative reason for the growth impairment. To further increase the potential applications of the RNAi-competent parasite, Ago2 expression was restricted to the liver stage using a stage-specific promoter. This transgenic line behavee indistinguishable from wild type and the expression of an AgoshRNA targeting GFP silenced fluorescence exclusively in late liver stages. In summary, PbAgo2 is a potent tool to modulate gene expression without the need to alter the genetic locus. In contrast to existing tools, PbAgo2 provides the option to target genes exclusively in a single life cycle stage, to multiplex different AgoshRNAs enabling the simultaneous knockdown of multiple genes, or to screen for phenotypes using a library of AgoshRNAs. This novel, RNAi-competent parasite line opens a wealth of new options to annotate genes in Plasmodium.