Exploring Trafficking Mechanisms During the Plasmodium Liver Stage

Abstract

Malaria is a devastating disease caused by the pathogen from the genus Plasmodium. Over half the world’s population lives at risk of infection every day. The parasite develops throughout a complex life cycle that involves both a mosquito vector and mammalian host. Once an infected female Anopheles mosquito takes a blood meal, the parasite is deposited into the skin of the mammalian host, where it rapidly enters the blood stream and migrates to the liver for the first stage of infection. Once in the liver, parasites asexually replicate and form blood-infective merozoites, which egress back into the blood stream. Afterwards merozoites cyclically invade, asexually replicate, and burst the host red blood cell, causing the clinical symptoms of malaria. The liver stage was only discovered about 50 years ago, largely because it is asymptomatic. Since then, researchers have sought to understand parasite biology and host-pathogen interactions at this essential stage. This dissertation aims to study liver stage infection by investigating how host proteins are trafficked to the parasite and how the parasite proteome changes during development. During the liver stage, the Plasmodium parasite develops within a parasitophorous vacuole (PV), surrounded by the PV membrane (PVM) that separates the parasite from the host cytosol. The PVM is the site for host-parasite interactions and is highly dynamic throughout the liver stage. Interestingly, some host proteins localize to the PVM and have been shown to be essential for parasite development. However, the mechanisms for host protein recruitment remain unresolved. Here, we found that the parasite relies on host endocytosis and exocytosis processes for protein recruitment and development. We identified the host SNARE protein VAMP3 as important for liver stage development. Using ultrastructure expansion microscopy and chemical inhibition of VAMP3 vesicle recycling, we show that VAMP3 vesicles fuse to the PVM. Overall, our study mechanistically reveals that host protein recruitment is linked to SNARE vesicle fusion. Liver stage P. berghei rapidly expand between 24 and 48 hours post infection. We sought to understand the parasite proteome throughout the liver stage to better understand their biology. Using the proximity labeling technique called biotinylation by antibody recognition, we labeled and enriched parasite proteins from fixed cells before identification by mass spectrometry. Interestingly, when we compared our data with RNA-seq, it suggests that liver stage parasites use translational repression programs to orchestrate their rapid development. In the future this methodology could be coupled with chemical biology techniques like noncanonical amino acids and thermal proteome profiling to resolve parasite protein dynamics and drug target identification. The host hepatocyte is broadly remodeled during liver stage infection, which is evident in RNA-seq datasets that show major transcriptional changes after invasion. The parasite manipulates host gene expression to down-regulate harmful pathways like cellular immunity and up-regulates processes beneficial for their development. This large reprogramming could be achieved through parasite effector proteins, which are well known in the blood stage of Plasmodium infection but have not been validated in the liver stage. We sought to lay the groundwork for the identification of putative effector proteins through the generation of cell lines that support proximity-dependent biotinylation in Plasmodium-infected host cells. Collectively, these studies investigate liver stage Plasmodium biology and its interactions with the host cell. Further, the tools and methods generated here can support future work to advance our understanding of liver stage biology and possible drug targets.