MA/MSc Internship for EUGLOH program

Keywords: malaria, transcription, ChIP

Internship Duration: 30/11/-1 - 30/11/-1

Malaria is caused by the unicellular eukaryotic parasite Plasmodium. Half a million people die of malaria, each year. Such high mortality is due to the existence of drug resistant parasites. It is thus urgent to find new drugs and new drug targets
The parasite life cycle is complex, involving two hosts - a human and a mosquito. The mosquito also serves as vector. The disease symptoms are correlated with the exponentially increasing waves of red blood cell (RBC) invasion and egress, in which parasite forms called schizonts rupture and release merozoites fit for a new round of RBC invasion. Alternatively, some schizonts (≈10%) enter the sexual pathway and differentiate into the sexual forms called gametocytes. Both pathways are indispensable for parasite survival. Continuous invasion of RBCs exponentially increases the number of parasites and differentiation into gametocytes ensures host-host transmission.
We have previously shown that the ApiAP2 transcription factor AP2-I of the human malaria parasite, Plasmodium falciparum, regulates transcription of several invasion genes (Santos et al., 2017). Another ApiAP2 protein called AP2-G activates transcription of gametocyte-specific genes (Kafsack et al., 2014). Transcription of ap2-g is tightly regulated. In the majority of cells, the ap2-g locus is transcriptionally silent due to binding of heterochromatin protein HP1 and repressive histone marks and, subsequently, AP2-G is not expressed, sexual genes are not transcribed and cells commit to asexual differentiation. In a small subset of cells, there is expression of a protein called GDV1 that destabilizes HP1 binding to ap2-g (Filarsky et al., 2018), triggering transcription of ap2-g and gametocyte formation.
ap2-g and gdv1 have been identified as AP2-I target genes by ChIP-Seq (Santos et al., 2017), suggesting that AP2-I regulates not only transcription of invasion genes but also of ap2-g and gdv1. In this project, the student will test this hypothesis by performing different experiments:
-mutate the AP2-I DNA motifs within the ap2-g and gdv1 promoters using CRISPR-Cas9 in AP2-I-GFP parasites
-use the mutant parasites to validate AP2-I binding to the promoters (by ChIP-qPCR)
-test whether in the mutant parasites, transcription of ap2-g and gdv1 is affected (by RT-qPCR)
At the end of the project, the student will have determined if AP2-I is the transcription factor regulating transcription of gametocyte master regulators. If AP2-I regulates simultaneously expression of invasion genes and of ap2-g and gdv1, it is the first identified parasite factor that if blocked, disrupts both asexual and sexual development and thus, cures the host and stops transmission.

-RT-qPCR
-ChIP-qPCR
-molecular cloning
-cell culture

Santos J. M., Josling G., Ross P., Joshi P., Orchard L, Campbell T., Schieler A., Cristea I. M., Llinás M. (2017) Red Blood Cell Invasion by the Malaria Parasite is Coordinated by the PfAP2-I Transcription Factor. Cell Host & Microbe 21, 731-41
Kafsack, B.F., Rovira-Graells, N., Clark, T.G., Bancells, C., Crowley, V.M., Campino, S.G., Williams, A.E., Drought, L.G., Kwiatkowski, D.P., Baker, D.A., et al. (2014). A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507, 248-252
Filarsky, M., Fraschka, S.A., Niederwieser, I., Brancucci, N.M.B., Carrington, E., Carrio, E., Moes, S., Jenoe, P., Bartfai, R., and Voss, T.S. (2018). GDV1 induces sexual com

• Pharmacy, Biology, Physics and Chemistry