Department of Microbiology and Immunology, Stanford University, Stanford, California, United States of America.
PLoS One. 2009 Dec 2;4(12):e8143. doi: 10.1371/journal.pone.0008143.
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 large number of target proteins in the parasite that are involved in at least two key steps: invasion and intracellular growth. This potent compound, the modified "click-chemistry" forms of it, and the resistant mutants should serve as useful tools to further study the processes of Toxoplasma early invasion, in general, and rhoptry secretion, in particular.
刚地弓形虫是顶复门的一种真核寄生虫，能够感染多种宿主细胞。在其主动侵袭过程中，它从离散的分泌细胞器：微线体、棒状体和致密颗粒中分泌蛋白质。虽然已经有许多棒状体蛋白被证明参与了与宿主细胞的重要相互作用，但对于任何弓形虫蛋白分泌到宿主细胞的机制知之甚少。我们使用了一种磷脂酶 A2s 的化学抑制剂，4-溴苯甲酰溴（4-BPB），来研究这种脂肪酶在弓形虫蛋白分泌中的作用。我们发现 4-BPB 是弓形虫侵袭中棒状体分泌的有效抑制剂。这种药物特异性地阻断了棒状体的分泌，但不阻断微线体的分泌，因此有效地表明这两个过程可以解耦。它影响寄生虫的运动和侵袭，但不影响附着或出芽。使用炔丙基或叠氮衍生物（所谓的点击化学衍生物）和一系列 4-BPB 抗性突变体，我们发现该药物在寄生虫中有大量的靶蛋白，这些靶蛋白至少参与了两个关键步骤：侵袭和细胞内生长。这种有效的化合物、其修饰的“点击化学”形式以及抗性突变体应作为进一步研究弓形虫早期侵袭过程的有用工具，特别是棒状体分泌过程。
