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.
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World Health Organization. World Malaria Report 2015 (World Health Organization, 2015).
Webster, W.A. & McFadden, G.I. From the genome to the phenome: tools to understand the basic biology of Plasmodium falciparum. J. Eukaryot. Microbiol. 61, 655–671 (2014).
de Koning-Ward, T.F., Gilson, P.R. & Crabb, B.S. Advances in molecular genetic systems in malaria. Nat. Rev. Microbiol. 13, 373–387 (2015).
Woodcroft, B.J., McMillan, P.J., Dekiwadia, C., Tilley, L. & Ralph, S.A. Determination of protein subcellular localization in apicomplexan parasites. Trends Parasitol. 28, 546–554 (2012).
Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
Robinson, M.S., Sahlender, D.A. & Foster, S.D. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev. Cell 18, 324–331 (2010).
Xu, T., Johnson, C.A., Gestwicki, J.E. & Kumar, A. Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization. Nat. Protoc. 5, 1831–1843 (2010).
Haruki, H., Nishikawa, J. & Laemmli, U.K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31, 925–932 (2008).
Jullien, N., Sampieri, F., Enjalbert, A. & Herman, J.P. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, e131 (2003).
Andenmatten, N. et al. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat. Methods 10, 125–127 (2013).
Collins, C.R. et al. Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Mol. Microbiol. 88, 687–701 (2013).
Fairhurst, R.M. Understanding artemisinin-resistant malaria: what a difference a year makes. Curr. Opin. Infect. Dis. 28, 417–425 (2015).
Szymczak, A.L. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).
Straimer, J. et al. Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nat. Methods 9, 993–998 (2012).
Wang, P., Wang, Q., Sims, P.F. & Hyde, J.E. Rapid positive selection of stable integrants following transfection of Plasmodium falciparum. Mol. Biochem. Parasitol. 123, 1–10 (2002).
Geda, P. et al. A small molecule-directed approach to control protein localization and function. Yeast 25, 577–594 (2008).
Patury, S., Geda, P., Dobry, C.J., Kumar, A. & Gestwicki, J.E. Conditional nuclear import and export of yeast proteins using a chemical inducer of dimerization. Cell Biochem. Biophys. 53, 127–134 (2009).
Papanikou, E., Day, K.J., Austin, J. & Glick, B.S. COPI selectively drives maturation of the early Golgi. eLife 4, e13232 (2015).
Dvorin, J.D. et al. A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science 328, 910–912 (2010).
Brancucci, N.M. et al. Heterochromatin protein 1 secures survival and transmission of malaria parasites. Cell Host Microbe 16, 165–176 (2014).
Elliott, D.A. et al. Four distinct pathways of hemoglobin uptake in the malaria parasite Plasmodium falciparum. Proc. Natl. Acad. Sci. USA 105, 2463–2468 (2008).
Poteryaev, D., Datta, S., Ackema, K., Zerial, M. & Spang, A. Identification of the switch in early-to-late endosome transition. Cell 141, 497–508 (2010).
Kremer, K. et al. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes. PLoS Pathog. 9, e1003213 (2013).
Spielmann, T. & Gilberger, T.W. Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways? Trends Parasitol. 26, 6–10 (2010).
Le Roch, K.G. et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503–1508 (2003).
Ariey, F. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2014).
Straimer, J. et al. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science 347, 428–431 (2015).
Witkowski, B. et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect. Dis. 13, 1043–1049 (2013).
Caro, F., Miller, M.G. & DeRisi, J.L. Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum. Malar. J. 11, 22 (2012).
Armstrong, C.M. & Goldberg, D.E. An FKBP destabilization domain modulates protein levels in Plasmodium falciparum. Nat. Methods 4, 1007–1009 (2007).
Jones, M.L. et al. A versatile strategy for rapid conditional genome engineering using loxP sites in a small synthetic intron in Plasmodium falciparum. Sci. Rep. 6, 21800 (2016).
Prommana, P. et al. Inducible knockdown of Plasmodium gene expression using the glmS ribozyme. PLoS One 8, e73783 (2013).
Ghorbal, M. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat. Biotechnol. 32, 819–821 (2014).
Boncompain, G. et al. Synchronization of secretory protein traffic in populations of cells. Nat. Methods 9, 493–498 (2012).
Varnai, P., Thyagarajan, B., Rohacs, T. & Balla, T. Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells. J. Cell Biol. 175, 377–382 (2006).
Birnbaum, J. et al. Selection linked integration (SLI) for endogenous gene tagging and knock sideways in Plasmodium falciparum parasites. Protocol Exchange http://dx.doi.org/10.1038/protex.2017.022 (2017).
Crabb, B.S. et al. Transfection of the human malaria parasite Plasmodium falciparum. Methods Mol. Biol. 270, 263–276 (2004).
Meyer, M. et al. Overexpression of differentially expressed genes identified in non-pathogenic and pathogenic Entamoeba histolytica clones allow identification of new pathogenicity factors involved in amoebic liver abscess formation. PLoS Pathog. 12, e1005853 (2016).
Heiber, A. et al. Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog. 9, e1003546 (2013).
Tóth, D.J. et al. Acute depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate impairs specific steps in endocytosis of the G-protein-coupled receptor. J. Cell Sci. 125, 2185–2197 (2012).
Hawthorne, P.L. et al. A novel Plasmodium falciparum ring stage protein, REX, is located in Maurer's clefts. Mol. Biochem. Parasitol. 136, 181–189 (2004).
Mesén-Ramírez, P. et al. Stable translocation intermediates jam global protein export in Plasmodium falciparum parasites and link the PTEX component EXP2 with translocation activity. PLoS Pathog. 12, e1005618 (2016).
Trager, W. & Jensen, J.B. Human malaria parasites in continuous culture. Science 193, 673–675 (1976).
Fidock, D.A. & Wellems, T.E. Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. Proc. Natl. Acad. Sci. USA 94, 10931–10936 (1997).
Moon, R.W. et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc. Natl. Acad. Sci. USA 110, 531–536 (2013).
Epp, C., Raskolnikov, D. & Deitsch, K.W. A regulatable transgene expression system for cultured Plasmodium falciparum parasites. Malar. J. 7, 86 (2008).
Malleret, B. et al. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development. Sci. Rep. 1, 118 (2011).
Grüring, C. & Spielmann, T. Imaging of live malaria blood stage parasites. Methods Enzymol. 506, 81–92 (2012).
Spielmann, T., Fergusen, D.J. & Beck, H.P. etramps, a new Plasmodium falciparum gene family coding for developmentally regulated and highly charged membrane proteins located at the parasite-host cell interface. Mol. Biol. Cell 14, 1529–1544 (2003).
We thank F. Kruse (BNITM) for cloning 2×FKBP into pARL-GFP and F. Sanchez-Roman (BNITM) for cloning FKBP into pMSRP6-mDHFR-mCherry. We are grateful to I. Bruchhaus (BNITM) for providing the plasmid pNC. We thank ARIAD Pharmaceuticals for providing plasmids encoding FKBP and FRB. We thank Jacobus Pharmaceuticals for WR99210. The following reagent was obtained through MR4/BEI Resources, NIAID, NIH: DSM1, MRA-1161. S.F., A.B.S. and E.J. gratefully acknowledge funding from the research training group GRK 1459 (German Research Foundation).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Graph showing time to integration for SLI versus conventional drug cycling using plasmids with Neomycin (NeoR) or Blasticidine resistance (BlaR) for SLI. Plasmids 1, 2, and 3: candidates #1, #2 and #8, respectively (Fig. 4); Plasmid 4 is CDPK5 (Fig. 2); plasmids 5 and 6 mediate expression of REX1-mDHFR-GFP and PFD0095c-mDHFR-GFP, respectively (Fig. S1). (b) Agarose gels with PCR products from genomic DNA of the parasite lines indicated above each gel. Primers used are as indicated in Fig. 1, demonstrating a product across the 5’ and 3’ integration junction (indicated as 5’ int and 3’ int, respectively) as well as quantitative absence of the original locus (‘original locus’). Absence of this band indicates that no parasites with the wild type locus remained in the parasite population. KI: knock in cell line; 3D7: wild type parental line. TGD: targeted gene disruption using SLI. Asterisks denote cell lines gernated with pSLIsandwich, leading to fusion of the target with the double tag (2xFKBP-GFP-2xFKBP), all others are 2xFKBP-GFP fusions. Fragment length of the marker is indicated once. For the Blasticidine SLI cell lines images showing the location of the endogenously GFP-tagged exported proteins are shown. DIC: differential interference contrast; DAPI: parasite nuclei; merge: merged red and green signal. Size bar: 5μm.
(a) Immunoblots with protein extracts of the cell lines shown in (Fig. 1b) demonstrate quantitative skipping. Expected sizes are 137, 100, 93, 123, 134 and 119 kDa, respectively (the skipped part would add 29 kDa for NeoR and 14 kDa for BlaR). Note that REX1 is known to show aberrant migration1. Detection was done using anti-GFP. Asterisk: frequently observed degradation product consisting of the GFP tag alone; hash: antibody independent ECL signal of hemoglobin. (b) Time to integration of plasmids into the genome plotted versus average expression values2 of the target. (c) Schematic of the principle for KS using the nucleus as site for mislocalisation. NLS, nuclear localisation signal; mCh, mCherry. (d) Proof of principle for rapalog induced localisation based on FKBP and FRB (one of 3 independent experiments): cytosolic mCherry-FKBP is re-localised to the mitochondrion carrying Mito-GFP-FRB in live P. falciparum parasites 1 h after addition of rapalog. DIC: differential interference contrast; DAPI: parasite nuclei; merge: merged red and green signal. Size bar: 5μm. (e) Quantification of mitochondrial recruitment of mCherry-FKBP after addition of rapalog indicated as proportion of mCherry fluorescence at the mitochondrion versus the cytoplasmic fluorescence normalised to the levels of Mito-GFP-FRB at these sites. n = 7, 8, 9, 9, and 8 cells for 0, 15, 30, 45 and 60 min, respectively. The quantification shown was done with images from two independent experiments. Central bars indicate the mean; error bars: SD.
1. Hawthorne, P. L. et al. A novel Plasmodium falciparum ring stage protein, REX, is located in Maurer's clefts. Mol Biochem Parasitol 136, 181-189 (2004).
2. Le Roch, K. G. et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-1508, doi:10.1126/science.1087025 (2003).
Shown are the FC growth curves over 5 days of 3 independent experiments for each cell line indicated. Day 0 represents the time when the culture was split into two dishes with ~0.1% parasitemia and then grown with and without rapalog (‘rapalog’ and ‘control’, respectively) to induce KS or diCre-based gene excision. Where applicable, the experiment shown in the main figure is marked with a cross. Top rows show wild type 3D7 parasites (WT) transfected with the mislocaliser, demonstrating no effect on growth of rapalog treatment or presence of the mislocaliser. Targets denoted with an asterisk carry the double tag (2xFKBP-GFP-2xFKBP), all others are 2xFKBP-GFP fusions. The type of mislocaliser used is indicated (1xNLS, 3xNLS, or Lyn). diCre indicates cell lines where addition of rapalog was used to induce diCre-based excision of the target gene.
Knock in cell lines of (a) PfMon1-2xFKBP-GFP and (b) PfArfGAP-2xFKBP-GFP expressing an mCherry tagged nuclear mislocaliser (MislocaliserN). Representative live cell images of rapalog treated and control cells (after 16 h) are shown next to FC growth curves (one representative of 3 independent experiments that are all shown in Fig. S3) and Giemsa stained blood smears of synchronised parasites for (a). A schematic of the protein is shown above each panel (not to scale), indicating the conserved domains. DIC: differential interference contrast; merge: merged red and green signal. Asterisk: food vacuole auto fluorescence. Size bar: 5μm.
(a) Schematic of the integration strategy for N-terminal tagging. The functional copy of Rab5a was recodonised (recod.) to prevent homologous recombination of this region. The first loxP site is in frame. Asterisks: stop codons; Small arrows labelled 1-5 indicate primers to verify integration and diCre-based gene excision by PCR. (b) PCRs using the primers indicated in (a) from gDNA of the cell lines indicated (diCre: 48 h after addition of rapalog): excised or non-excised locus (1+5), locus before excision (1+2), integration into the endogenous locus (3+4) and intact endogenous locus (1+4). (c) Representative fluorescence images of live GFP-2xFKBP-Rab5a expressing P. falciparum parasites at different stages (trophozoite through to schizont, top to bottom). DIC: differential interference contrast; DAPI: parasite nuclei. Size bar: 5μm.
Representative fluorescence images of live P. falciparum parasites for the cell lines indicated (asterisks denote cell lines where a 2xFKBP-GFP-2xFKBP tag was used, all others are 2xFKBP-GFP fusions). Where applicable, on rapalog (KS) and control are shown. DIC: differential interference contrast; DAPI: parasite nuclei; merge: merged red and green signal; MislocaliserN: nuclear mislocaliser; MislocaliserP: PPM mislocaliser. Asterisk: food vacuole autofluorescence. Size bar: 5μm. Where applicable, FC growth curves are shown (one representative of 3 independent experiments that are all shown in Fig. S3). n.d.: no FC growth assay done. For the undetectable candidate 14, a nuclear and a PPM mislocaliser was used without effect on growth. This may indicate that this protein is not essential for parasite survival, as also indicated by SLI-TGD (Fig. 4a). However, due to the lack of detectable GFP signal, efficient mislocalisation could not be verified. For candidate #12 a 1xNLS mislocaliser expressed under the hsp86 promoter in a plasmid with the yDHODH resistance was tested (see materials and methods). Due to the strong overexpression and the comparably weak nuclear targeting signal, the mislocaliser does not appear concentrated in the nucleus. However, as the target contains 4 FKBP domains (double 2xFKBP), addition of rapalog results in more nuclear localisation signals on the target than on the mislocaliser alone, causing efficient transfer of the target to the nucleus.
Supplementary Figure 7 KS kinetics and slow selection of cells expressing low levels of mislocalizer.
(a) FC curve of a growth assay for candidate 11 extended to 9 days. (b) Fluorescence images of the mislocaliser signal (mCherry) taken with identical settings from live parasites on day 8 from the growth experiment in (a). (c) Quantification of the fluorescence intensity (average per cell) in the control and the parasites grown under rapalog (n = 22 and 21 cells for control and rapalog, respectively) taken on day 8 of the growth experiment shown in (a). (d) Comparison of the time for mislocalisation to occur in a cytosolic protein present in foci (Rab5a), a cytosolic protein with a dispersed distribution [PF3D7_0218200 (candidate 18)] and two nuclear proteins [PF3D7_0205600 (candidate 11) and PF3D7_0210200 (candidate 13)] after addition of rapalog after the time indicated above the panels. Arrowheads: non-mislocalised protein; asterisks: food vacuole autofluorescence; DIC: differential interference contrast; DAPI: parasite nuclei; merge D/G: merge DAPI and GFP; merge M/G: merge mislocaliser and GFP; size bar: 5μm. Pie charts for GFP-Rab5a and PF3D7_0218200-GFP show proportion of cells where the GFP signal fully co-localised with the mislocaliser, suggesting full mislocalisastion (black) or cells where GFP signal did not fully co-localise, suggesting incomplete mislocalisation (grey); n = 18, 20, and 23 cells for GFP-Rab5a and 30, 22, and 20 cells for PF3D7_0218200-GFP, for 1 h, 4 h, and 8 h, respectively. For the nuclear proteins (PF3D7_0205600-GFP and PF3D7_0210200-GFP) incomplete mislocalisation was defined as focal or dispersed GFP signal in the area occupied by the DAPI nuclear stain. Pie charts show proportion of cells with incomplete mislocalisation (grey) versus cells with complete mislocalisation (black). For PF3D7_0205600-GFP n = 24, 19, 39, and 52 cells for 1 h, 4 h, 8 h, and 16 h, respectively; for PF3D7_0210200-GFP n = 26, 20, and 16 cells, for 1 h, 4 h, 8 h, and 16 h, respectively. The results in (a-c) and in (d) each show one of two independently replicated experiments.
Asterisks: stop codons; 2A: T2A skip peptide; arrows: promoters.
(a) Schematic of integration strategy using SLI for N-terminal fusion and floxing the active gene copy. Genomic integration of the plasmid leads to the expression of a second resistance marker (yDHODH, yeast dihydroorotate dehydrogenase), that is fused to the expression cassette via two skip peptides (2A), leading to separate expression products. This enables SLI. C580Y indicates the mutated alternative gene copy conferring Artemisinin resistance that was used on a second plasmid to generate a resistant knock in cell line. Small arrows labelled 1-4 show primers for PCRs used to confirm integration. (b) Western blot with parasite protein extracts of GFP-2xFKBP-Kelch13 (138 kDa) probed with rabbit anti-GFP and 2xFKBP-GFP-2xFKBP-Kelch13 (165 kDa) probed with anti-FKBP (the sandwiched GFP is not well detected with anti-GFP). (c) Maximum intensity projections of 3D reconstructions of live Bodipy-TR-C5-ceramide (Bodipy-ceramide) labelled GFP-2xFKBP-Kelch13 cells (images of individual focal planes acquired by confocal microscopy). DIC: differential interference contrast. Arrowheads show Kelch13 foci close to the parasite food vacuole; arrows show foci not close to the food vacuole, e.g. at the parasite plasma membrane. (d) KS with GFP-2xFKBP-Kelch13. Left: representative live cell images. Middle: FC growth assay (one representative of 3 experiments that are all shown in Fig. S3). Right, quantification of protein mislocalised (n = 10 cells; error bars show SD). DIC: differential interference contrast; merge: merged red and green signal. Size bars: 5μm.
(a) Removal of the kelch13 gene using diCre-based inducible excision (control: untreated; +rapalog: induction of diCre dimerisation). Left, representative live cell images acquired with identical settings. Size bar: 5μm. The arrowhead denotes faint left over signal. a.i.: after induction of diCre-based gene-excision. A quantification of fluorescence of foci was done for one of four independent experiments and is shown below (n = 14, 18, 10 and 23 foci for the control and 17, 26, 5 and 7 foci for the rapalog sample, the median and p values are indicated, two-sample Wilcoxon rank-sum (Mann-Whitney) test). Live cycle stages in these cultures are indicated for each day. Rings are shown on day 3 and 4 of the control for comparison with the culture on rapalog which only contained rings at these time points. Right, growth assay (one of 3 independent assays, all FC curves shown in Fig. S3). (b) Giemsa smear shows presence of condensed rings 5 days after induction of diCre to remove kelch13.
FC plots highlighting the parasitemia (% of all RBC) of ring stages (Rings) versus later stage parasites (Trophs/Schizonts) during a 5 day growth experiment for the following 3 cell lines: GFP-2xFKBP-Kelch13 (Kelch13) expressing diCre, 2xFKBP-GFP-2xFKBP-Kelch13 (Kelch13wt*) expressing the 1xNLS mislocaliser and 2xFKBP-GFP-2xFKBP-Kelch13 (Kelch13wt*) expressing the 1xNLS mislocaliser and diCre after induction of gene removal by diCre, KS or KS and diCre-based gene excision, respectively. Days after induction by addition of rapalog (days a.i.) are indicated to the left. For each cell line one of the 3 independent replicas of the FC growth assays (Fig. S3) was used for the plots shown in this figure.
Supplementary Figure 12 Simultaneous diCre-based gene excision and KS with 2×FKBP-GFP-2×FKBP-Kelch13.
(a) representative FC growth curve (one of 3 independent experiments that are all shown in Fig. S3) of 2xFKBP-GFP-2xFKBP-Kelch13 expressing the 1xNLS mislocaliser and diCre, permitting simultaneous diCre-based gene excision and KS. (b) fluorescence images of the 2xFKBP-GFP-2xFKBP-Kelch13 parasites (Kelch13wt*) one and two days after induction (a.i.) of diCre-based gene excision and KS (the images shown are from one of four independent experiments). Acquisition times for the green channels were the same for all images. DIC, differential interference contrast; merge, merged red and green signal. The asterisk denotes food vacuole autofluorescence. Size bar, 5μm.
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Birnbaum, J., Flemming, S., Reichard, N. et al. A genetic system to study Plasmodium falciparum protein function. Nat Methods 14, 450–456 (2017). https://doi.org/10.1038/nmeth.4223