The liver stage of the malaria parasite that develops inside hepatocytes of the mammalian host has been the most challenging stage of the complex Plasmodium parasite life cycle to study. Yet, the liver stage is the most promising target for protective vaccine development and also a good preventive drug target because it occurs without any clinical symptoms and precedes the pathogenic blood stages. We have recently made great advances in gaining experimental access to liver stages through the use of fluorescent activated cell sorting (FACS) to isolate liver stage-infected hepatocytes. This technique allows us to routinely obtain liver stages from mouse infections and from in vitro infected hepatocytes. This advance enables proteomic analysis for the first time. Since Plasmodium is an intracellular parasite enclosed by a vacuole, we hypothesize that the crucial parasite proteins for host modulation must be located either on the vacuole surface or exported beyond the vacuole to the host. To have a better understanding of the biology of the liver stage parasite and its interaction with its host hepatocyte, we will carry out proteomic analysis of the isolated infected hepatocytes. By identifying potential parasite proteins exported to the parasitophorous vacuole membrane (PVM) surface and host hepatocyte cytoplasm (i.e., exportome) we hope to identify Plasmodium proteins that are important for vacuole formation and growth and survival in the host hepatocyte during the liver stage development.
The specific aims are: 1) Identify the proteome of Plasmodium liver stages using tandem mass spectrometry analysis to identify proteins from purified infected hepatocytes at early, mid and late stages of development;and 2) Identify potential exported parasite proteins targeted to the liver stage PVM or in the host cytoplasm through proteomic analysis of proteins from the PVM and cytoplasmic proteins from lysates obtained from liver stage- infected hepatoma cells.
The proposed exploratory research will present the first comprehensive data on the proteome of the Malaria liver stages. The identification of the subset of parasite proteins that are exported to the parasite parasitophorous vacuolar membrane and host hepatocyte will identify proteins that may be critical for liver stage development and will provide new potential drug and vaccine targets.
