During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parasitophorous vacuole to safely reside within the cell, while it is partitioned from host cell defense mechanisms. From within this safe niche, parasites sabotage multiple host cell systems, including gene expression, apoptosis, and intracellular immune recognition, by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. The translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes the proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined that GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins, including MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell c-Myc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen.IMPORTANCE Approximately one-third of humans are infected with the parasite Toxoplasma gondii Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. Toxoplasma replication requires that it enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

中文翻译：

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刚地弓形虫分泌的酸性磷酸酶结构域的GRA44是感染细胞中c-Myc诱导所必需的。

在宿主细胞入侵期间，真核病原体弓形虫形成了寄生虫液泡，可以安全地驻留在细胞内，同时它与宿主细胞防御机制分开。在这种安全的环境中，寄生虫通过分泌大量的效应蛋白来破坏多个宿主细胞系统，包括基因表达，凋亡和细胞内免疫识别。研究用于主动宿主细胞操纵相互作用的许多寄生虫蛋白是激酶。效应子从寄生虫液泡向宿主细胞的转运是由推定的translocon复合物介导的，该复合物包括蛋白质MYR1，MYR2和MYR3。尚不清楚其他蛋白质是否参与该推定的转座子的结构或调控。我们发现分泌蛋白GRA44 含有推定的酸性磷酸酶结构域，与该复合物的成员相互作用，是效应子分泌下游的宿主细胞效应所必需的。我们已经确定，在与分泌途径的原生动物蛋白酶靶向的序列具有同源性的区域中加工了GRA44，并且两个主要切割片段都被分泌到了寄生虫的液泡中。免疫沉淀实验表明GRA44与大量分泌的蛋白质（包括MYR1）相互作用。重要的是，条件性敲低GRA44导致缺乏宿主细胞c-Myc上调，这类似于当translocon复合物的成员被基因破坏时所见的表型。从而，推定的酸性磷酸酶GRA44对于弓形虫感染期间宿主细胞的改变至关重要，并且与弓形虫成功作为细胞内病原体所依赖的translocon复合物有关。重要大约三分之一的人感染了寄生虫弓形虫弓形虫感染可以导致免疫系统受损或受到抑制的人患有严重疾病。另外，怀孕期间的感染对发育中的胎儿具有重大的健康风险。靶向该寄生虫的药物是有限的，具有明显的副作用，并且不能靶向所有疾病阶段。因此，彻底了解寄生虫如何在宿主内传播对于发现新型治疗靶标至关重要。弓形虫复制需要它进入被感染生物的细胞。为了在细胞内的环境中生存，弓形虫分泌大量的蛋白质，这些蛋白质劫持了许多重要的细胞功能。这些弓形虫蛋白如何从寄生虫转移到宿主细胞中尚不清楚。我们的工作表明，假定的磷酸酶GRA44是负责此过程的蛋白质复合物的一部分。