Cell cycle checkpoints and checkpoint kinases in the apicomplexan parasites Plasmodium and Toxoplasma

Malaria affects millions of people annually and causes many fatalities. Most cases of severe malaria are caused by the *Plasmodium falciparum* parasite. *P. falciparum* is transmitted from Anopheles mosquitoes to humans, where parasites infect hepatocytes and then erythrocytes. Intra-erythrocytic stages cause the clinical symptoms of malaria and are mainly treated by the front-line drug artemisinin. Since the late 2000’s there has been a rise in artemisinin-resistant parasites in the Greater Mekong Subregion, and similar phenotypes have now been detected in some regions of Africa, proving this is becoming a more pressing issue. DNA repair mechanisms and particularly cell cycle checkpoint mechanisms in *P. falciparum* are not well characterised. A greater understanding of these is required, especially as artemisinin- resistant parasites have a dormancy phenotype that resembles a cell cycle checkpoint. Elucidating these mechanisms and identifying potential checkpoint kinases is, therefore, essential to understanding how *P. falciparum* resists this frontline drug. Checkpoints are essential to maintaining growth and genetic stability in many other eukaryotic cells. Whether these checkpoints are as essential in apicomplexan parasites, which have very divergent forms of replication, needed to be explored. Phosphatidylinositol 3-kinase-related kinases (PIKKs) are responsible for checkpoint control in model eukaryotes. Members of this protein family are missing in *Plasmodium*, but some remain in the close relative *Toxoplasma gondii*. A suite of experiments were conducted to investigate these potential checkpoints, including cellular assays on the effect of PIKK-inhibiting drugs on *P. falciparum* and *T. gondii*; and exploration of whether artemisinin-resistant or -sensitive *P. falciparum* lines could be resistant or hypersensitivity to PIKK inhibitors. We also explored the potential dual role of phosphatidylinositol 3-kinase, *Pf*PI3K, as a checkpoint kinase, through several experimental avenues such as a complementation project, recombinant protein expression, and tracking of the *Pf*PI3K protein *in-vivo* via tagging or antibody generation. In *T. gondii*, we characterised the putative homologues of the key PIKK kinases from human cells, ATR and ATM. Neither proved to be essential, but they did exhibit checkpoint protein activity. We then attempted a complementation of *Pf*PI3K in inducible knockout lines of these proteins. Greater understanding of how these parasites operate and control their unusual cell cycles is essential to improve treatment methods and potential patient outcomes, especially in the context of antimalarial drug resistance, and in such biologically intriguing and early-diverging organisms.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Johnson, Monique

Advisor dc:contributor.advisor

• Merrick, Catherine

• Checkpoint kinases
• Plasmodium
• Toxoplasma

Licence

• https://www.repository.cam.ac.uk/bitstreams/08a39649-60bc-4f92-8b4e-08ce474ab360/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.112970

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375209