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Role of perforin-like proteins and phospholipases in malaria parasite egress

Periodic Reporting for period 1 - MalariaEgress (Role of perforin-like proteins and phospholipases in malaria parasite egress)

Reporting period: 2018-01-01 to 2019-12-31

Malaria is one of the world’s deadliest diseases that infects around 228 million people worldwide. Around 400,000 people die of malaria every year and most of them are children under five years of age in malaria-endemic sub-Saharan Africa. Malaria is caused by a single-celled parasite, Plasmodium, and is most commonly spread via bite from a female Anopheles mosquito. Of the five Plasmodium species that infect humans, Plasmodium falciparum is responsible for the majority of malaria related deaths. Malaria is preventable and treatable but progress towards controlling the disease is threatened by emergence of drug resistance in the parasite. The clinical manifestations of the disease arise from the parasite’s blood stages when it goes through multiple cycles of asexual replication and destroys human red blood cells (RBCs) in the process. All effective antimalarial drugs target the asexual blood stages, which therefore make good chemotherapeutic targets for developing antimalarials. To this end, we need a proper understanding of how malaria parasites propagate within host erythrocytes.
Malaria parasites grow and replicate asexually within a parasitophorous vacuole in host RBCs. As the parasite grows, it has to form membranous structures for nutrient uptake from the host and maintain its plasma membrane and the parasitophorous vacuole membrane (PVM). As the parasite starts to replicate, it also has to develop membranes for various cellular compartments like the nucleus, apicoplast and mitochondrion. Thus, the malaria parasite has to produce and maintain a substantial amount of membranes and this results in a ~6-fold increase in phospholipid (PL) content in infected RBCs. The drastic membrane dynamics requires a finely tuned lipid metabolism that involves lipids being synthesized de novo or scavenged from the host and then also modified, transported and degraded by the parasite.
At the end of each cycle of replication, malaria parasites exit the host cell in a coordinated manner, a crucial process known as egress, to invade fresh RBCs. Egress involves a rapid sequence of events that results in the rupture of the PVM and the host RBC membrane (RBCM). We know that egress is triggered by protein kinase G-dependent discharge of a subtilisin protease, SUB1, into the PV lumen where SUB1 cleaves and activates several effector molecules. One such effector molecule, SERA6, a serine protease, has been shown to cleave host spectrin in the RBC cytoskeleton to bring about the final event of RBCM rupture. However, effector molecules involved in the preceding steps of PVM rupture and RBCM poration are unknown.
Phospholipases (PLases) are lipolytic enzymes that target and cleave PLs. They play key roles in lipid metabolism and mediate various cell functions such as membrane synthesis, degradation and signaling. The parasite produces several PLases throughout its asexual life cycle, but little is known about the specific roles each of these enzymes play. Owing to their membranolytic activity, it is also possible that one of the PLases causes PVM rupture during egress.
In this project, we selected and studied the role of four phospholipases by inducing knockout of their genes in P. falciparum. We have also worked towards devising a medium-throughput inducible genetic screen to identify phospholipases that play a role during parasite egress. Our study shows that PLases play vital roles in membrane dynamics in the malaria parasite and is required for several processes throughout the parasite’s asexual life cycle.
We used state-of-the-art DiCre-mediated conditional gene knockout technology to knockout four candidate phospholipases. We used biochemical, cell imaging, electron microscopy and lipidomics approaches to characterize their functions.
Main exploitable results-
1) A Plasmodial phospholipase- lecithin: cholesterol acyltransferase (PfLCAT) is required for efficient blood-stage egress in the parasite but not essential for parasite survival.
2) A patatin-like phospholipase A2 (PfPLPA2) is required for proper formation of mitochondria but not essential for parasite survival.
3) Phosphoinositide-specific phospholipase C (PfPI-PLC) is essential for schizont stage growth in the parasite.
4) A parasite glycerophosphodiesterase (PfGDPD) is essential for trophozoite growth in the parasite.
5) Proof-of-principle experiments show that a DiCre-based inducible genetic screen is workable once the different components (under development) are assembled.
The results from this study will be disseminated through three publications that are currently under preparation. Part of the results were presented to malaria researchers at the BioMalPar conference in Heidelberg in 2019.
There are 23 putative phospholipases in P. falciparum, but their functions have not been fully elucidated. We have carried out a detailed functional analysis of four members of this class of enzymes and show that each plays a role at a particular point in the parasite’s 48 hr asexual life cycle. Our results show that synthesis and breakdown of phospholipids are well-regulated and are vital for the parasite to survive and propagate. The enzymes and pathways involved in phospholipid metabolism are likely attractive drug targets.
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