CRISPR/Cas9-engineered inducible gametocyte producer lines as a novel tool for basic and applied research onPlasmodium falciparummalaria transmission stages

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 to other human hosts via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialized cells. Here, we engineered marker-free P. falciparum inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes. Through targeted overexpression of the sexual commitment factor GDV1, iGP lines consistently achieve sexual commitment rates of 75% and produce gametocytes that are infectious to mosquitoes. Subsequent tagging of a nucleoporin allowed us to visualize marked nuclear transformations during gametocytogenesis and demonstrates that further genetic engineering of iGP lines is an invaluable tool for the targeted exploration of gametocyte biology. We believe the iGP approach developed here opens up unprecedented opportunities that will expedite future basic and applied research on P. falciparum transmission stages.

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

“…To obtain the NF54/PKAc cOE parasite line, the pD_pkac_cOE donor plasmid was generated in several cloning steps using Gibson assembly reactions. First, a pD_cOE_DD precursor plasmid was generated using a two-fragment Gibson assembly joining (1) the plasmid backbone including the glp3 -specific 5’ and 3’ HRs amplified from pD_ cg6_cam-gdv1-gfp-glmS 82 (primers glp3_F and glp3_R) and (2) the cam 5’-gfpdd-hrp2 3’ sequence amplified from pHcamGFP-DD 52 (primers cam_F and hrp2_R). Second, the subsequent precursor plasmid pD_cOE_glmS was generated by performing a Gibson assembly using two fragments (1) the pD_cOE_DD precursor plasmid digested using SalI and AgeI and the (2) glmS sequence amplified from pD_ cg6_cam-gdv1-gfp-glmS 82 (primers glmS_F and glmS_R).…”

“…First, a pD_cOE_DD precursor plasmid was generated using a two-fragment Gibson assembly joining (1) the plasmid backbone including the glp3 -specific 5’ and 3’ HRs amplified from pD_ cg6_cam-gdv1-gfp-glmS 82 (primers glp3_F and glp3_R) and (2) the cam 5’-gfpdd-hrp2 3’ sequence amplified from pHcamGFP-DD 52 (primers cam_F and hrp2_R). Second, the subsequent precursor plasmid pD_cOE_glmS was generated by performing a Gibson assembly using two fragments (1) the pD_cOE_DD precursor plasmid digested using SalI and AgeI and the (2) glmS sequence amplified from pD_ cg6_cam-gdv1-gfp-glmS 82 (primers glmS_F and glmS_R). To insert a new cloning site and generate the next precursor plasmid pD_cOE, another Gibson assembly reaction was performed joining (1) the pD_cOE_glmS plasmid digested using BamHI and NotI and (2) annealed complementary oligonucleotides clon_F and clon_R.…”

“…The final pD_pkac_cOE plasmid was generated by assembling two Gibson fragments: (1) the BamHI- and SpeI- digested pD_cOE plasmid and (2) the pfpkac sequence amplified from NF54 gDNA using primers pka_F and pka_R. The pBF_gC- cg6 suicide plasmid that was co-transfected with pD_pkac_cOE to generate this NF54/PKAc cOE parasite line encodes the sgRNA targeting the glp3 locus 82 . To obtain the NF54/PKAcT189V cOE parasite line, the pD_pkacT189V_cOE donor plasmid was generated in a Gibson assembly joining three fragments: (1) the BamHI- and SpeI- digested pD_cOE plasmid; (2) the 5’ fragment of the pfpkac sequence containing the single point mutation resulting in the T189V amino acid change (primers pka_F and T189V_R); and (3) the 3’fragment of the pfpkac sequence overlapping with the 5’ fragment and coding for the same amino acid change (primers T189V_F and pka_R).…”

Ultrastructure expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a microtubule organisation centre coordinating mitosis with axoneme assembly

“…The fine ultrastructural architecture of each stage has been previously thoroughly investigated by detailed conventional EM [4] or more recently by Serial Block Face Scanning EM [21]. Here we combined U-ExM with bulk proteome [22,23] and α/β tubulin labelling to image the intracellular development of gametocytes using the iGP2 line [24].…”

“…falciparum culture. In vitro cultures to produce P. falciparum gametocytes using the iGP2 line was performed as described in [24]. Briefly, the parasite line was grown in human erythrocytes in RPMI-1640 medium with glutamine (Gibco), 0.2% sodium bicarbonate, 25 mM HEPES, 0.2% glucose, 5% human serum, and 0.1% Albumax II (Life Technologies).…”