Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

Abstract

Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.

Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

“…This involved transfecting pRhopH2-HAglmS into P. falciparum that when correctly integrated into the rhopH2 locus, would lead to incorporation of a triple hemagglutinin (HA) and single strep II tag at the C-terminus of RhopH2 and the glucosamine (GlcN)-inducible glmS ribozyme (Prommana et al, 2013) within its 3' untranslated region (UTR) (Figure 1a). Diagnostic PCR of transfectants resistant to WR99210 selection after three rounds of drug cycling confirmed that transgenic parasites, termed PfRhopH2-HAglmS, harbored the expected integration event (Figure 1b).…”

An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes

“…4a and Supplementary Fig. 4a)35. We similarly produced inducible knockdown lines for two additional Pf EMP1B-interacting proteins, PF3D7_1226900 and PF3D7_1024800.…”

PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

“…We utilized conditional translational inhibition via the glmS ribozyme that is activated by glucosamine (GlcN) (Prommana et al, 2013). Using CRISPR/Cas9, we added a triple-V5 tag to the C-terminus of PfClpP and PfClpR followed by the glmS sequence (Figure 4A).…”