CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research

Abstract

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

Fig. 1. Description of the inducible gametocyte producer line 3D7/iGP.

a Schematic of the disrupted cg6 locus carrying a single inducible GDV1-GFP-DD expression cassette. The 5′ and 3′ homology regions used for CRISPR/Cas9-based transgene insertion are shown in orange. b Schematic of the in vitro culture protocol used to quantify sexual commitment rates (SCRs). Synchronous 3D7/iGP ring stage parasites are split at 0–16 hpi and Shield-1 is added to one half of the population to trigger GDV1-GFP-DD expression. SCRs are quantified by determining the proportion of early stage I gametocytes in the total iRBC progeny 36–44 hpi (day 2 of gametocytogenesis) by α-Pfs16 IFAs combined with DAPI staining. Asexual parasites are depicted in grey, sexually committed parasites and gametocytes are depicted in purple. asS/scS, asexual/sexually committed schizont; asR/scR, asexual/sexually committed ring stage; T, trophozoite; I–V, gametocyte stages I–V; D1–D10, days 1–10 of gametocyte maturation. Gen 1/2, generation 1/2. c α-Pfs16 IFA images illustrating the high proportion of early gametocytes in the progeny of Shield-1-treated 3D7/iGP parasites. The white arrow highlights a Pfs16-negative schizont. Nuclei were stained with DAPI. DIC, differential interference contrast. Images are representative of three independent experiments. Scale bar, 10 µm. d Proportion of Pfs16-positive iRBCs (SCRs) in the progeny of 3D7/iGP treated with three different Shield-1 concentrations and the untreated control (−Shield-1) (mean ± SD, n = three biologically independent experiments; two experiments for parasites treated with 337.5 nM Shield-1). Closed circles represent data points for individual experiments (>156 DAPI-positive cells counted per experiment).

Fig. 2. Description of the inducible gametocyte producer lines NF54/iGP1 and NF54/iGP2.

a Schematics of the disrupted cg6 locus carrying a single inducible GDV1-GFP-DD-glmS or GDV1-GFP-glmS expression cassette in NF54/iGP1 or NF54/iGP2, respectively. The 5′ and 3′ homology regions used for CRISPR/Cas9-based transgene insertion are shown in orange. b Proportion of Pfs16-positive iRBCs (SCRs) in the progeny of NF54/iGP1 and NF54/iGP2 cultured under noninducing or inducing conditions (mean ± SD, n = three biologically independent experiments). Closed circles represent data points for individual experiments (≥391 DAPI-positive cells counted per experiment). c Schematic of the in vitro culture protocol used to obtain pure NF54/iGP2 stage V gametocyte populations. GlcN is removed from the culture medium of synchronous ring stage parasites at 0–16 hpi to trigger expression of GDV1-GFP. After schizont rupture and merozoite invasion, gametocyte maturation proceeds for >10 days. Asexual parasites are depicted in grey, sexually committed parasites and gametocytes are depicted in purple. asS/scS, asexual/sexually committed schizont; asR/scR, asexual/sexually committed ring stage; T, trophozoite; I–V, gametocyte stages I–V; D1–D10, days 1–10 of gametocyte maturation. Gen 1/2, generation 1/2. d Images of Giemsa-stained NF54/iGP2 gametocyte cultures, acquired on day 1 (asexual/sexually committed ring stages), day 6 (stage III gametocytes), and day 11 (stage V gametocytes). Images are representative of three independent experiments. Scale bar, 20 µm. Parasitemias determined from three independent induction experiments are shown on the right (≥2068 RBCs counted per experiment). e Proportion of Pfs16-positive iRBCs (SCRs) in the progeny of NF54/iGP1_D8 and NF54/iGP2_E9 parasites cultured under noninducing or inducing conditions and of NF54 wt control parasites (mean ± SD, n = three biologically independent experiments). Closed circles represent data points for individual experiments (≥143 DAPI-positive cells counted per experiment). f Western blot showing expression of GDV1-GFP (MW=99.1 kDa) and GDV1-GFP-DD (MW=111.3 kDa) in NF54/iGP2_E9 and NF54/iGP1_D8 schizonts (34–42 hpi), respectively. PfHP1 (MW = 31 kDa) served as a control to compare the relative numbers of nuclei loaded per lane. The results are representative of two independent experiments. g Sex ratios of NF54/iGP2_E9 stage V gametocytes obtained via GDV1-GFP overexpression (−GlcN) or via induction of sexual commitment using mFA medium (+GlcN/mFA), as quantified from α-Pfg377 IFAs (mean ± SD, n = three biologically independent experiments). Closed circles represent data points for individual experiments (≥192 gametocytes scored per experiment). Sex ratios were compared using a paired two-tailed Student’s t test (p value indicated above the graph).

Fig. 3. NF54/iGP1_D8 and NF54/iGP2_E9 gametocytes complete their life cycle in the mosquito vector and produce infectious sporozoites.

a NF54/iGP1_D8 (orange), NF54/iGP2_E9 (blue), and NF54 wt control stage V gametocytes (green) were fed to female Anopheles stephensi mosquitoes on day 10, 13, and 14 of gametocytogenesis in two independent SMFA experiments. The violin plots show the distribution of the number of oocysts detected in each of the 20 mosquitoes dissected per feed, with open circles and triangles representing data from SMFA replicates 1 and 2, respectively (left y axis). The median (thick red line) and upper and lower quartiles (thin red lines) are indicated. Closed red circles represent the mean oocyst prevalence (number of infected mosquitoes) determined for each of the two replicate feeds (right y axis). NF54 wt day 10 and day 13 gametocytes were only included in SMFA replicate 1, and for SMFA replicate 2, only 10 mosquitoes infected with NF54 wt day 14 gametocytes have been dissected. b Mean number of salivary gland sporozoites per oocyst (left y axis) and per mosquito (closed red circles; right y axis) 17 days after infection with NF54/iGP1_D8 (orange), NF54/iGP2_E9 (blue), and NF54 wt control day 14 gametocytes (green) (SMFA replicate 2 data). Values represent the results from a single experiment (≥26 mosquitoes dissected per infected batch). c Confocal microscopy IFA images showing intracellular parasites after infection of primary human hepatocytes with NF54/iGP1_D8, NF54/iGP2_E9, and NF54 wt control sporozoites. Parasites were stained with α-PfHSP70 (cytosol; red) and α-PfEXP2 antibodies (parasitophorous vacuolar membrane; purple). α-GFP antibodies were used to test for potential ectopic expression of GDV1 in liver stages. Nuclei were stained with DAPI. Images are representative of a single experiment. Scale bar, 18 µm.

Fig. 4. Visualisation of nuclear pore distribution in NF54/iGP2_NUP313-mSc asexual blood stage parasites and gametocytes.

a Schematic maps of the disrupted cg6 locus carrying a single inducible GDV1-GFP-glmS expression cassette and the tagged nup313 locus in the double-transgenic NF54/iGP2_NUP313-mSc line are shown on top. The 5′ and 3′ homology regions used for CRISPR/Cas9-based genome editing are shown in orange. Live-cell fluorescence microscopy images showing the localisation of NUP313-mScarlet (red) in asexual blood stage parasites. ER/LR, early/late ring stage; T, trophozoite; ES/LS, early/late schizont; M, merozoite. DIC, differential interference contrast. Nuclei were stained with DAPI. Images are representative of three biologically independent experiments. Scale bar, 5 µm. White frames refer to the magnified view presented in the rightmost images (scale bar, 2 µm). b Live-cell fluorescence microscopy images showing the localisation of NUP313-mScarlet (red) in stage I to V gametocytes. Lateral extensions of the nucleus away from Hoechst-stained bulk chromatin are highlighted by yellow arrowheads. I–V, stage I to V gametocytes. DIC, differential interference contrast. Nuclei were stained with Hoechst (blue). Images are representative of four biologically independent experiments. Scale bar, 5 µm. White frames refer to the magnified view presented in the rightmost images (scale bar, 2 µm).

Fig. 5. Nuclei in stage II–V gametocytes undergo marked morphological transformations.

Schematic maps of the disrupted cg6 locus carrying a single inducible GDV1-GFP-glmS expression cassette and the tagged nup313 locus in double-transgenic NF54/iGP2_NUP313-mSc line are shown on top. The 5′ and 3′ homology regions used for CRISPR/Cas9-based genome editing are shown in orange. Live-cell fluorescence microscopy images showing the localisation of NUP313-mScarlet (red) in stage II to V gametocytes. Lateral extensions (yellow arrowheads) or rounded expansions (pink arrowheads) of the nucleus away from Hoechst-stained bulk chromatin and separate NUP313-mScarlet-delineated regions enclosing (white arrowheads) or devoid of Hoechst-stained bulk chromatin (blue arrowhead) are highlighted. II–V, stage II to V gametocytes. DIC, differential interference contrast. Nuclei were stained with Hoechst. Images are representative of four biologically independent experiments. Scale bar, 5 µm. White frames refer to the magnified view presented in the rightmost images (scale bar, 2 µm).

Fig. 6. Scheme depicting the simple induction protocol for the routine mass production of NF54/iGP gametocytes and potential applications for future research.

Addition of Shield-1/removal of GlcN (NF54/iGP1) or removal of GlcN (NF54/iGP2) from a synchronous ring stage culture triggers sexual commitment in trophozoites and produces progeny consisting of up to 75% sexual ring stage parasites. Addition of 50 mM GlcNAc to the culture medium for the next six days eliminates the remaining asexual parasites. Expected numbers of parasite-infected RBCs (#) and percent parasitaemia (% P) of total (black letters), asexual (grey letters), and sexual (red letters) parasites in the progeny routinely obtained from a 10 ml culture at 5% haematocrit (HC) and 1.5% starting parasitaemia are indicated. Asexual parasites are depicted in grey, sexually committed parasites and gametocytes are depicted in purple (females) and green (males). asS/scS asexual/sexually committed schizont, asR/scR asexual/sexually committed ring stage, T trophozoite, I–V gametocyte stages I–V, D1–D10 days 1–10 of gametocyte maturation, G gametes, Z zygote, Ok ookinete, Oc oocyst, Sp sporozoites, hepS intrahepatic schizont. Possible applications of NF54/iGP lines for basic, applied, and translational research on P. falciparum gametocytes and mosquito-stage parasites are listed below the schematic.