Doerig, C. | bioRxiv Science

bioRxiv2018

Validation of the protein kinase PfCLK3 as a multi-stage cross species malarial drug target

The requirement for next generation anti-malarials to be both curative and transmission blockers necessitate the identification of molecular pathways essential for viability of both asexual and sexual parasite life stages. Here we identify a selective inhibitor to the Plasmodium falciparum protein kinase PfCLK3 which we use in combination with chemogenetics, whole genome sequencing and transcriptomics to validate PfCLK3 as a druggable target acting at multiple parasite life stages. Consistent with the proposed role of PfCLK3 as a regulator of RNA splicing, inhibition results in the down-regulation of >400 genes essential for parasite survival. Through this mechanism, blocking PfCLK3 activity not only results in rapid killing of asexual blood stage parasites but is also effective on sporozoites and gametocytes as well as showing parasiticidal activity in all Plasmodium species tested. Hence, our data establishes PfCLK3 as a target with the potential to deliver both symptomatic treatment and transmission blocking in malaria.

microbiology↗

bioRxiv2018

Inducing controlled cell cycle arrest and re-entry during asexual proliferation of Plasmodium falciparum malaria parasites.

The life cycle of the malaria parasite Plasmodium falciparum is tightly regulated, oscillating between stages of intense proliferation and quiescence. Cyclic 48-hour asexual replication of Plasmodium is markedly different from cell division in higher eukaryotes, and mechanistically poorly understood. Here, we report tight synchronisation of malaria parasites during the early phases of the cell cycle by exposure to DL--difluoromethylornithine (DFMO), which results in the depletion of polyamines. This induces an inescapable cell cycle arrest in G1 (~15 hours post-invasion) by blocking G1/S transition. Cell cycle-arrested parasites enter a quiescent G0-like state but, upon addition of exogenous polyamines, re-initiate their cell cycle in a coordinated fashion. This ability to halt malaria parasites at a specific point in their cell cycle, and to subsequently trigger re-entry into the cell cycle, provides a valuable framework to investigate cell cycle regulation in these parasites. We therefore used gene expression analyses to show that re-entry into the cell cycle involves expression of Ca2+-sensitive (cdpk4 and pk2) and mitotic kinases (nima and ark2), with deregulation of the pre-replicative complex associated with expression of pk2. Changes in gene expression could be driven through transcription factors MYB1 and two ApiAP2 family members. This new approach to parasite synchronisation therefore expands our currently limited toolkit to investigate cell cycle regulation in malaria parasites.

cell biology↗