Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA.
Center for Tropical and Emerging Global Diseases, University of Georgia, Indianapolis, IN, USA.
Cell Microbiol. 2017 Sep;19(9). doi: 10.1111/cmi.12749. Epub 2017 May 17.
Toxoplasma gondii is an obligate intracellular apicomplexan parasite with high seroprevalence in humans. Repeated lytic cycles of invasion, replication, and egress drive both the propagation and the virulence of this parasite. Key steps in this cycle, including invasion and egress, depend on tightly regulated calcium fluxes and, although many of the calcium-dependent effectors have been identified, the factors that detect and regulate the calcium fluxes are mostly unknown. To address this knowledge gap, we used a forward genetic approach to isolate mutants resistant to extracellular exposure to the calcium ionophore A23187. Through whole genome sequencing and complementation, we have determined that a nonsense mutation in a previously uncharacterised protein is responsible for the ionophore resistance of one of the mutants. The complete loss of this protein recapitulates the resistance phenotype and importantly shows defects in calcium regulation and in the timing of egress. The affected protein, GRA41, localises to the dense granules and is secreted into the parasitophorous vacuole where it associates with the tubulovesicular network. Our findings support a connection between the tubulovesicular network and ion homeostasis within the parasite, and thus a novel role for the vacuole of this important pathogen.
刚地弓形虫是一种专性细胞内顶复门寄生虫，在人类中具有高血清流行率。入侵、复制和逸出的反复裂解循环既驱动了寄生虫的传播，也驱动了其毒力。该循环中的关键步骤，包括入侵和逸出，依赖于严格调控的钙离子流，尽管已经鉴定出许多依赖钙离子的效应物，但检测和调节钙离子流的因素大多未知。为了解决这一知识空白，我们使用正向遗传学方法分离出对细胞外暴露于钙离子载体 A23187 具有抗性的突变体。通过全基因组测序和互补，我们确定了一个以前未被描述的蛋白中的无义突变是一个突变体对离子载体产生抗性的原因。该蛋白的完全缺失再现了抗性表型，并且重要的是显示出在钙离子调节和逸出时间方面的缺陷。受影响的蛋白 GRA41 定位于致密颗粒并分泌到吞噬空泡中，在那里它与管状囊泡网络相关联。我们的发现支持了管状囊泡网络与寄生虫内部离子动态平衡之间的联系，因此为这种重要病原体的空泡提供了一个新的作用。
