4-bromophenacyl bromide specifically inhibits rhoptry secretion during toxoplasma invasion溴化4-bromophenacyl专门抑制rhoptry分泌在弓形虫入侵.pdfVIP

4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion 1 1 2 3 1 Sandeep Ravindran , Melissa B. Lodoen , Steven H. L. Verhelst , Matthew Bogyo , John C. Boothroyd * 1 Department of Microbiology and Immunology, Stanford University, Stanford, California, United States of America, 2 Center for Integrative Protein Science Munich, Chemistry of Biopolymers, Technical University Munich, Freising, Germany, 3 Department of Pathology and Microbiology and Immunology, Stanford University, Stanford, California, United States of America Abstract Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa that is able to infect a wide variety of host cells. During its active invasion process it secretes proteins from discrete secretory organelles: the micronemes, rhoptries and dense granules. Although a number of rhoptry proteins have been shown to be involved in important interactions with the host cell, very little is known about the mechanism of secretion of any Toxoplasma protein into the host cell. We used a chemical inhibitor of phospholipase A2s, 4-bromophenacyl bromide (4-BPB), to look at the role of such lipases in the secretion of Toxoplasma proteins. We found that 4-BPB was a potent inhibitor of rhoptry secretion in Toxoplasma invasion. This drug specifically blocked rhoptry secretion but not microneme secretion, thus effectively showing that the two processes can be de-coupled. It affected parasite motility and invasion, but not attachment or egress. Using propargyl- or azido-derivatives of the drug (so-called click chemistry derivatives) and a series of 4-BPB-resistant mutants, we found that the drug has a very la