Investigating ubiquitination-regulated cell cycle events underpinning malaria transmission

Malaria is caused by the unicellular pathogen Plasmodium that threatens around 400 million people globally and results in over 0.5 million deaths annually, thereby continuing to be a major public health problem, and urgently requiring new therapeutics. Completion of the malaria parasite's complex lifecycle requires a mammalian host where it causes disease, as well as a mosquito vector responsible for spread of the disease. Consequently, effective elimination of malaria will require both curative and transmission blocking strategies.

Similar to other eukaryotes, Plasmodium replicates it's genome to divide, proliferate and spread. However, several unusual characteristics of parasite genome replication can be exploited to combat the parasite, especially in stages crucial for parasite transmission. Parasite transmission to the mosquito is initiated by sexual male and female gametocytes. Upon experiencing the mosquito environment, the male gametocyte undergoes three rounds of genome replication (called mitosis) with an incredible speed of 10 minutes to form sperm-like gametes that fuse with the female cell. The fertilised zygote then undergoes further genome replication (called meiosis) to develop into a motile ookinete that is responsible for infecting mosquitoes. So how is mitosis in gametocytes and meiosis in zygotes regulated?
We discovered that many proteins in gametocytes and zygotes are dynamically modified by the small protein, ubiquitin. Ubiquitin is reversibly attached to proteins to modulate their fate, including their stability, cellular localisation and level of activity, suggesting these reversible marks could play a key role during parasite genome replication. Importantly, an eraser of ubiquitin marks, Plasmodium USP7 (ubiquitin specific protease 7) is crucial for the parasite to complete both mitosis and meiosis. Since it is not experimentally possible to study meiosis in the human malaria parasite (P. falciparum), we will exploit the highly conserved rodent malaria model (P. berghei), to examine how USP7 regulates genome replication both during mitosis and meiosis.

In this proposal we will uncover how USP7 prepares the parasite for initiation of DNA replication during transmission stages. Using state-of-the-art proteomics and microscopy techniques, we will identify partners and responders of USP7 and also determine how the enzyme activity and structure of parasite USP7 is divergent from its host's equivalent. Our findings will be influential in establishing platforms to screen for pharmacological USP7 inhibitors. Moreover, identifying how USP7 orchestrates both mitosis and meiosis will be useful in the development of improved therapeutic strategies that target and block multiple steps in parasite transmission.

Genome replication is fundamental to proliferation of all living cells. Although the core DNA replication machinery is conserved across higher eukaryotes, cell cycle events in evolutionarily divergent organisms, such as Plasmodium (the malaria parasite), are regulated by unusual replication and chromatin dynamics. Plasmodium proliferates using mitosis in the vertebrate host, while both mitotic and meiotic genome replication are crucial for transmission stages in the mosquito. During transmission, sexual precursor cells or male gametocytes can achieve incredible speeds of DNA replication to complete three rounds of genome replication within just 10 minutes to generate motile gametes that fertilize female cells to form zygotes. Zygotes undergo meiotic replication that can take up to three hours. Although several signalling pathways regulated by phosphorylation have been identified to regulate DNA replication, we still know little about how the parasite prepares the DNA and chromatin architecture prior to duplication of the genome. We recently discovered that an eraser of ubiquitin marks, the deubiquitinase USP7 (ubiquitin specific protease 7), to be a central regulator for initiating DNA replication in both mitosis and meiosis. This is particularly exciting because removal of USP7 completely blocks parasite transmission and deubiquitinase enzymes are very tractable to inhibition by small pharmaceuticals. Understanding the interplay between USP7 and the signalling networks that regulate mitosis and meiosis will reveal new targets and strategies for transmission blocking. Using advanced proteomic, genetic and imaging tools we will (i) reveal the divergent domain and enzyme activity of parasite USP7 (ii) uncover partners and effectors of USP7 that control mitosis and meiosis (iii) functionally analyse USP7 substrate repertoires to reveal how dynamic ubiquitination mediated by USP7 underpins diverse genome replication events in malaria transmission stages.