: Successful parasitism by the obligate intracellular Toxoplasma gondii is critically dependent on efficient nutrient scavenging and subversion of diverse host functions. Access to nutrients and cellular functions within the host cytoplasm is restricted by the parasitophorous vacuole membrane (PVM), a parasite modified structure that serves as the boundary defining the replication permissive niche. Activities at the PVM are critical in nutrient acquisition, structural integrity and the manipulation of host signaling cascades, including potentially the immune response to the parasite. While the PVM is involved in many facets of infection the molecular basis for these activities remains elusive. The inability to purify the PVM has prevented detailed biochemical analyses of these activities. In this proposal we aim to characterize the proteome of the T. gondii PVM using mass spectroscopic approaches coupled with novel polyclonal antisera as affinity reagents. In addition to conventional mass spectroscopic techniques we will apply multi-dimensional protein identification technology (MudPit) which permits the analysis of complex protein mixtures. These approaches to define the PVM proteome are made feasible by recent advances in the mass spectrometric identification of proteins, open access to the T. gondii genome databases and bioinformatic algorithms to analyze the data. The culmination of this approach is to establish the foundation for the characterization of activities in the PVM by defining its protein composition. These approaches will likely lead to the identification of potentially novel chemotherapeutic targets for Toxoplasma and other apicomplexan including Cryptosporidium parvum and hepatic stages of Plasmodia. ? ? ?
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