Rome Michael E, Beck Josh R, Turetzky Jay M, Webster Paul, Bradley Peter J
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles CA 90095-1489, USA.
Infect Immun. 2008 Nov;76(11):4865-75. doi: 10.1128/IAI.00782-08. Epub 2008 Sep 2.
Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.
刚地弓形虫是一种专性细胞内寄生虫，它寄生于宿主细胞的细胞质中，存在于一种独特的膜结合泡囊中，即被称为寄生泡（PV）的结构。寄生虫周围的膜会被致密颗粒重塑，致密颗粒是一种分泌细胞器，它会将一系列蛋白质释放到泡囊和寄生泡膜（PVM）中。目前仅鉴定出了致密颗粒中的一小部分蛋白质成分，对于它们在感染过程中的作用以及在被感染宿主细胞内的运输方式知之甚少。在本报告中，我们鉴定出一种新的分泌型致密颗粒蛋白GRA14，并表明它靶向于泡囊内被称为泡内网络的膜结构以及寄生虫周围的泡膜。我们破坏了GRA14，并利用基因敲除菌株表明，在与野生型寄生虫的共感染实验中，GRA14可以在泡囊之间转移。我们还表明，GRA14在PVM中具有意想不到的拓扑结构，其C末端面向宿主细胞质，N末端面向泡腔。这些发现对于GRA蛋白运输到其最终目的地以及对宿主-寄生虫界面处GRA蛋白功能域的预期都具有重要意义。
