From antigenic variation to cell cycle progression: a functional analysis of the putative "var" gene regulator PfSET10 and its interaction partners in "Plasmodium falciparum"

Plasmodium falciparum causes the most devastating form of human malaria. Its remarkable replication potential is crucial to overcoming the complex life cycle bottlenecks. During blood-stage infection, parasites undergo repeated rounds of red blood cell (RBC) invasion and asexual replication that rapidly increase the parasite load in the human host. To avoid parasite clearance by the adaptive immune system, P. falciparum employs antigenic variation of surface antigens, such as P. falciparum erythrocyte membrane protein 1 (PfEMP1). Furthermore, PfEMP1 medi-ates cytoadherence of infected RBCs to vascular endothelium, which on the one hand prevents their passage through and removal by the spleen and on the other hand contributes strongly to malaria pathogenesis. Hence, the massive parasite replication combined with antigenic variation is crucial to parasite survival and transmission, and at the same time a major cause for severe disease outcomes.
PfEMP1 is encoded by 60 var gene paralogs, which are expressed in a mutually exclusive man-ner. var gene activation and switching are associated with nuclear repositioning to a specific perinuclear site termed var gene expression site (VES). However, the molecular details of the underlying regulatory mechanisms have yet to be entirely understood. To date, PfSET10 re-mains the only protein presumed to exclusively localise to the VES. Hence, during my PhD studies, I set out to scrutinize the role of PfSET10 and further describe its role in antigenic varia-tion. Additionally, I aimed to identify and characterise additional proteins associated with the VES. In the first study, I could convincingly rule out that PfSET10 regulates var gene expres-sion, thereby correcting the long-standing model of var gene regulation. The results from co-immunoprecipitation and in vivo proximity-dependent biotinylation experiments using PfSET10 as bait provide the foundation for the second study, where I conducted functional analyses of putative PfSET10 interaction partners. Here, the conditional knockout of one of these factors (Pf3D7_1322300) prevents further rounds of genome replication beyond the first S-phase dur-ing schizogony and male gametogenesis. This unique loss-of-function phenotype highlights that Pf3D7_1322300 is an essential and novel component of the parasite's replication machinery and provides the first evidence for an intra-S-phase checkpoint-like activities in P. falciparum. Moreover, we found that the PfSET10 complex interacts with the cohesin complex at the peri-centromeric region, thereby, our study provides the foundation for the intriguing possibility that the VES may be tethered via an unknown mechanism to the CP or the centromeric cluster. In the third study, we investigated the three Aurora-related kinase of malaria parasites and gained valuable insight into their expression dynamics and subcellular localisation to mitotic structures and cell division machinery during schizogony and gametocytogenesis. Collectively, the work presented here provides fascinating new insight into the molecular mechanisms underlying the control of cell cycle progression and var gene regulation, offering new perspectives for future research.