A genetic system to study Plasmodium falciparum protein function

Abstract

Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

“…To determine whether this was the case in P. falciparum , we attempted to knock out the GP1 gene using the newly developed selection‐linked integration for targeted gene disruption strategy (SLI‐TGD). This method provides greatly increased efficiency in the selection of parasites with genomic integrations (Birnbaum et al, ). Despite numerous attempts, we were unable to delete GP1 (data not shown).…”

“…To attempt the KO of GP1 and GP2, we used the recently described selection‐linked integration for targeted gene disruption (SLI‐TGD) strategy (Birnbaum et al, ). We generated the pSLI‐TGD‐GP1 vector by amplifying, on 3D7 P. falciparum gDNA, a fragment of 497pb at the 5′ extremity of the GP1 gene and cloned it into NotI‐MluI restriction sites of the pSLI‐TGD vector.…”

The malaria parasite Plasmodium falciparum Sortilin is essential for merozoite formation and apical complex biogenesis

“…However, a recent report in which PfRab5A was conditionally inactivated showed that parasites could successfully mature to the schizont stage, but interestingly, they were unable to egress. Unfortunately, the status of the apical complex and the IMC in these parasites were not investigated (Birnbaum et al, ). The late endosomal marker Rab7 and members of the retromer complex have also been localised in P. falciparum blood stages and shown to be distinct from the Golgi apparatus (Krai et al, ).…”

Structure-based identification and functional characterization of an essential lipocalin in the malaria parasite Plasmodium falciparum

“…Having shown that PfLCN is indeed a lipocalin based on its structure, we next determined its localization by endogenously tagging PfLCN with green fluorescent protein (GFP) using the selection-linked integration (SLI) system (Birnbaum et al, 2017). We additionally introduced a GlmS ribozyme (Prommana et al, 2013) sequence upstream of the 3' untranslated region that allows inducible knockdown of protein expression upon addition of glucosamine to the parasite culture medium ( Figure 3A).…”

“…In order to test if PfLCN is essential for blood stage proliferation, we first tried to functionally inactivate it by targeted-gene disruption using the SLI system (Birnbaum et al, 2017). However, repeated attempts failed to obtain parasites carrying the correctly integrated plasmid, indicating that the gene is likely essential for parasite development.…”