Proteomic interrogation of the parasite vacuole using autoSTOMP

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Project Summary Toxoplasma gondii (Tg) is an incurable, obligate intracellular protozoan parasite that naturally infects human and rodent hosts for life. Toxoplasma’s extraordinary ability to persist inside almost any nucleated cell depends on an intact parasite vacuole membrane (PVM). Secreted parasite effectors localized to the PVM interface subvert cell-intrinsic immune detection and import nutrients for growth. However, biochemical tools have not been available to identify the complete sets of host and parasite components recruited to the PVM. To recognize and clear Tg the host relies on two complementary cell autonomous immunity pathways. The interferon-y regulated GTPases (IRG) system detects the PVM as foreign and Toll-like receptors (TLRs) prime inflammasome cytosolic sensor recognition of Tg. Sub-cellular localization of the IRG system is a major point of regulation, however, the precise mechanism of PVM recognition, Tg clearance and control of host cell fate downstream of the IRG and TLR pathways are unclear. We hypothesize that immune recognition at the PVM controls parasite fate and host cell survival. To identify the critical regulators of host and parasite survival at the PVM we have developed a novel proteomics technology called automated spatially targeted optical micro proteomics or autoSTOMP. AutoSTOMP uses the confocal microscope to image the PVM and attach photo- activatable affinity purification tags to all PVM proteins so that they can be precipitated and identified by liquid chromatography and mass spectrometry (LC-MS). This tool allows us to easily compare PVM localized proteins across the three canonical types of Tg that differ in virulence by several logs. Here autoSTOMP will be used to understand how immune stimulation regulates protein recruitment to the PVM and controls the fate of Tg and host cells.