Aim 1 Progress:
Aiming to overcome the difficulty of isolating early stage sexual parasites, the gametocytes (morphologically similar to the asexual stages during the first 36 hours after invasion), a novel tool was designed to induce the expression of promiscuous biotin ligase A enzyme (BirA*) on the surface of the RBC infected with early stage gametocytes from 14 hours after the erythrocyte invasion and then use the streptavidin affinity technology to isolate the labelled parasites. The expression of BirA* was detected at the expected parasite stages near the RBC membrane in the genetically modified parasite line. However, the number of parasites expressing BirA* was very low and biotinilation of the infected RBC surface was not detected.
Aim 2 Progress:
Given the impossibility to use affinity purification using the tool from aim1 to do proteomic/phosphoproteomic analysis to address signaling pathways important during early sexual development, a collaboration has been established with Matthias Marti group at the University of Glasgow that has identified a molecule in human serum that actively supresses gametocytogenesis. The identification of the signaling pathways during sexual commitment and development by comparing the phosphoproteome of induced, non-induced and induced but inhibited parasites is currently being done. This approach will allow the identification of phosphorylation events that occur during induction of gametocyte development and signaling pathways.
Aim 3 Progress:
Progression through the five different morphological stages of sexual development (gametocytogenesis) and gamete activation (a process that occurs when mature stage V gametocytes are engorged by the mosquito in the blood meal) was addressed in 10 kinase knockout (KO) lines. Only PfTKL2 kinase KO (characterized now for the first time regarding gametocyte/gamete development) has shown a clear defect in sexual development. No mutations were detected on the ApiAP2G gene (a transcription factor essential from the very early steps of sexual development) suggesting that the gametocyteless phenotype observed in TKL2 KO was likely due to a potential role of the kinase in sexual commitment or early gametocyte development. To further characterize the PfTKL2 KO phenotype, new Plasmodium falciparum lines containing Cre recombinase were generated, 3D7A::DiCre and NF54::DiCre, and used to make a conditional KO of PfTKL2. DiCre-mediated recombination and excision, induced in the presence of rapamycin (Rap), allows for a tightly controllable conditional genetic system. Aiming to address the role of PfTKL2 during sexual development, the parasites were treated either with Rap or DMSO (control). Unlike observed with the original PfTKL2 KO, there was no difference between the PfTKL2 DMSO and Rap induced KO parasites regarding the percentage of sexual induction and gametocyte development nor in the number of female and male gametes. Aiming to further explore if PfTKL2 has a role during transmission, a PfTKL2 conditional KO has been generated in NF54 parasites (the line used to perform experiments through mosquitoes) and is currently being used to address PfTKL2 role during mosquito and liver infection.
At the same time the ten kinases KO lines were being tested, Pfeik2 kinase KO line, previously characterized to produce viable gametocytes and gametes, was used as a positive control. However, unlike expected, Pfeik2 KO line showed a clear defect in producing gametocytes. No mutations were detected in the ApiAP2G gene and PfGEXP05 (expressed from 14h post invasion of sexually committed parasites independently of ApiAP2G regulation) was used to identify sexual parasites. Results suggest that the block happens before PfGEXP05 expression and therefore, this is the first line with a clear defect in sexual commitment or early development, likely independent of ApiAP2G regulatory pathway. RNA sequencing (RNA-Seq) analysis comparing asexual and sexual parasites of 3D7A (wild type line) and 2 clones of the Pfeik2 kinase KO has been done in collaboration with The Sanger Institute, allowing the identification of a list of genes down-regulated, including PfGEXP05, independently of ApiAP2G transcriptional regulation. Furthermore, gdv1 gene, previously published to abolish gametocyte formation when knocked out, has been sequenced and a stop mutation has been identified. The same mutation was also identified in the original PfTKL2 KO line, strongly suggesting that the mutation in gdv1 could be responsible for abolishment of gametocyte formation. The identified mutation was introduced in gdv1 locus of a healthy NF54 line and is currently being used to address gdv1 role and interaction partners involved in Plasmodium falciparum sexual regulatory pathways.
The work developed has allowed identification of potentially novel players involved in Plasmodium falciparum sexual commitment/early development regulatory pathways, as well as the generation of a novel and invaluable tool to conditionally KO genes of interest. All the results obtained will be published, after peer reviewing and available to the scientific community.
