Some conditions apply: Systems for studying Plasmodium falciparum protein function

Abstract

Malaria, caused by infection with Plasmodium parasites, remains a significant global health concern. For decades, genetic intractability and limited tools hindered our ability to study essential proteins and pathways in Plasmodium falciparum, the parasite associated with the most severe malaria cases. However, recent years have seen major leaps forward in the ability to genetically manipulate P. falciparum parasites and conditionally control protein expression/function. The conditional knockdown systems used in P. falciparum target all 3 components of the central dogma, allowing researchers to conditionally control gene expression, translation, and protein function. Here, we review some of the common knockdown systems that have been adapted or developed for use in P. falciparum. Much of the work done using conditional knockdown approaches has been performed in asexual, blood-stage parasites, but we also highlight their uses in other parts of the life cycle and discuss new ways of applying these systems outside of the intraerythrocytic stages. With the use of these tools, the field’s understanding of parasite biology is ever increasing, and promising new pathways for antimalarial drug development are being discovered.

Gliding motility of Plasmodium merozoites

“…For instance, although our work shows that the inhibitor U73122, but not its inactive analog, U73343, inhibits signaling pathways also predicted to be required for motility; in other systems, the effects of both compounds have been shown to be identical, suggesting off-target activity ( 52 ). Therefore, future work should include targeted genetic approaches such as conditional knockout studies ablating signaling enzymes—something which is now readily achievable for Plasmodium blood stages in both P. knowlesi and P. falciparum ( 53 ).…”

Methods Used to Investigate the Plasmodium falciparum Digestive Vacuole

“…In order to functionally assess proteins that are essential to the blood stages, conditional regulatory systems are required. For reviews on genetic approaches used throughout Plasmodium research we direct readers to ( de Koning-Ward et al., 2015 ; Kudyba et al., 2021 ; Okombo et al., 2021 ). A number of these systems now exist and include regulation at the DNA, transcript, and protein level.…”

Recent Advances in Molecular Genetic Tools for Babesia

“…To study the protein function, protein level could be manipulated by inducing premature degradation by fusing protein to destabilizing domain or translocation of the target protein by a method called knock sideways, KS [62,63]. The advantage of targeting the protein of interest is the fast action of this system that could be leveraged for studying the rapid biological process [64]. We have validated FK506-binding protein (FKBP)-based destabilizing domain (DD) by fusing GFP with DD (Figure 3C, unpublished data).…”

“…DD could be stabilized by the addition of Shield 1 and in the absence of Shield 1, the target protein degradation will be promoted via the proteasome. The applicability of the DD system for Babesia requires further investigation; however, this system is not suitable for the membrane or secreted proteins that are not accessible to the proteasome in the parasite cytoplasm [64,65]. KS which initially called anchor-away is based on conditional tethering of the protein of interest by rapamycin-dependent dimerization where target protein is fused with FKBP and additionally, FRB is fused to a protein with different cellular localization called mislocalizer [63,66].…”

“…Conditional deletion of a target gene using dimerizable Cre recombinase (DiCre) has been established for Plasmodium spp. and was shown to be efficient for several targets in the in vitro and in vivo models [64,65,68]. This system has two compartments-DiCre, in which its inactive two proteins are fused upon rapamycin addition, and a short targeting sequence called loxP.…”