Abstract
During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro-cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P.berghei. IMPORTANCE Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In Plasmodium parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent P.berghei parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.
Highlights
During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells
Within hepatocytes and red blood cells (RBCs), Plasmodium parasites reside within a specialized compartment referred to as the parasitophorous vacuole (PV) and are surrounded by the PV membrane (PVM), which is initially formed by invagination of the host cell membrane (HCM) during the process of invasion
Other prominent parasitophorous vacuole membrane (PVM) proteins are the components of the Plasmodium translocon of exported proteins (PTEX), mediating the export of proteins into the host cell [12], and proteins belonging to the family of the early transcribed membrane proteins (ETRAMPs) [13]
Summary
During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; only a limited number of proteins of this membrane have been identified so far. A limited number of PVM proteins have been identified so far, and the functions of most of these remain elusive (reviewed in references 3 and 4) These include the earliest known PVM protein, exported protein 1 (EXP1), which contains a classical N-terminal signal peptide and is inserted into the PVM of blood and liver stage parasites with its transmembrane domain, whereby the C terminus faces the host cell cytoplasm [5,6,7,8]. Other prominent PVM proteins are the components of the Plasmodium translocon of exported proteins (PTEX), mediating the export of proteins into the host cell [12], and proteins belonging to the family of the early transcribed membrane proteins (ETRAMPs) [13]
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