A single-cell approach to study epigenetic regulation of Plasmodium knowlesi phenotypes: red blood cell invasion and gametocyte differentiation processes.
Development of a novel single cell technology to investigate epigenetic mechanisms in biological processes in P. vivax parasites. P. knowlesi is used for protocol development, it will be later used for P. vivax field samples since they have a close genetic relationship.
Transcriptional regulation in P. vivax is still poorly understood, although it plays an important role in key biological processes such as red blood cell invasion and gametocyte conversion. As transcription factors only play a minor role in transcriptional control, it is hypothesized that epigenetic regulation through histone modifications, just like in P. falciparum, play an important part. Until now, investigation of epigenetics in P. vivax was challenging due to the lack of long-term in vitro culture, which restricted research to low parasitaemia patient samples. With the arrival of single-cell sequencing technologies, it has become feasible to not only sequence low input samples, but also study them at the individual cell level, an important prerequisite to study epigenetics, as clonal populations show different expression patterns. We will measure both expression and chromatin conformation from the same cells, using a recently developed kit. We propose to use in vitro cultivated P. knowlesi, which is evolutionary and genetically close to P. vivax, to test parameters and optimize the protocol that will be later used for P. vivax field samples. Furthermore, biological insights will be gained on the transcriptional control of P. knowlesi red blood cell invasion process and gametocyte conversion, two processes which are expected to be epigenetically regulated. Those can provide a basis to investigate this in P. vivax and compare processes between both species.