CRISPR/Cas9-engineered inducible gametocyte producer lines as a novel tool for basic and applied research on Plasmodium falciparum malaria 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.

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum

“…Using the stronger detergent DDM did not lead to detection of more proteins or assembled complexes (Supplementary Table 1) and was consequently omitted in favour of digitonin solubilization for gametocyte samples. To overcome the challenge of obtaining sufficient gametocyte material, we used a recently established inducible gametocyte producer line (NF54/iGP2) (Boltryk et al, in revision 27 ) that allows for the synchronous mass production of GCT through conditional overexpression of gametocyte development 1 (GDV1), an activator of sexual commitment 28 . We observed no marked differences in proteome or morphology for ABS and GCT stages between the initial samples prepared with wild-type NF54 (GCT1Da, ABS1Ma, ABS1Da) and all remaining samples prepared with NF54/iGP2 parasites (Supplementary Table 1).…”

Investigation of heterochromatin protein 1 function in the malaria parasitePlasmodium falciparumusing a conditional domain deletion and swapping approach

“…Irrespective of this uncertainty, our findings strongly suggest that PbHP1 retained the capacity to interact with GDV1 and that this interaction evolved based on evolutionary conserved features of HP1 orthologs in malaria parasites. In light of this conclusion, it may be interesting to explore if ectopic expression of GDV1 could be used as an experimental tool for the induction of high sexual conversion rates in P. berghei , as recently reported for P. falciparum (59, 61).…”

“…In addition to virulence gene families, HP1 also silences the gene encoding AP2-G, the master transcriptional regulator of gametocytogenesis in malaria parasites (29, 30, 49, 57–59). AP2-G is a member of the ApiAP2 family of putative transcription factors of apicomplexan parasites (60).…”

Investigation of Heterochromatin Protein 1 Function in the Malaria Parasite Plasmodium falciparum Using a Conditional Domain Deletion and Swapping Approach

“…Irrespective of this uncertainty, our findings strongly suggest that PbHP1 retained the capacity to interact with GDV1 and that this interaction evolved based on evolutionarily conserved features of HP1 orthologs in malaria parasites. In light of this conclusion, it may be interesting to explore whether ectopic expression of GDV1 can be used as an experimental tool for the induction of high sexual conversion rates in P. berghei, as was recently reported for P. falciparum (58,60).…”