A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma.
A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma.
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DOI：
10.1371/journal.ppat.1003049
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
Arrizabalaga G
中科院分区：
文献类型：
--
作者：
Garrison E;Treeck M;Ehret E;Butz H;Garbuz T;Oswald BP;Settles M;Boothroyd J;Arrizabalaga G
从宿主细胞中排出是专性细胞内寄生虫弓形虫生物学中的关键和高度调节的步骤。主动外出依赖于钙流，似乎是逃避免疫系统攻击的关键步骤，并可能使寄生虫穿梭进入适当的细胞进入宿主的大脑。先前的遗传筛选已经产生了在离子载体诱导的外出和离子载体诱导的死亡中都有缺陷的突变体。使用一个突变体的全基因组测序和随后的所有突变体的分析，从这些屏幕上，我们发现，值得注意的是，四个独立的突变体在同一个基因，TgCDPK3，编码钙依赖性蛋白激酶的错义突变。预测所有四种突变都会改变TgCDPK3的关键区域，并且这通过对每种突变的重组形式的生物化学研究得到证实。通过互补，我们证实了TgCDPK3在快速诱导寄生虫排出中的关键作用，并且我们确定TgCDPK3对小鼠脑中潜伏期的形成至关重要。TgCDPK3的基因敲除证实了这种激酶在寄生虫排出中的关键作用和在裂解周期中的非必需作用。 弓形虫是一种专性细胞内寄生虫，是人类重要的病原体，因此进出细胞是其生存的关键。弓形虫离开细胞的过程被称为出口，由钙流控制，并可由离子载体触发。在体内，从宿主细胞的快速排出已被确定为逃避先天免疫系统攻击的手段。在分子水平上，这种寄生虫中的钙依赖性事件部分地由植物样钙依赖性激酶调节，所述植物样钙依赖性激酶与人类激酶没有同源性，因此是理想的药物靶标。在这项研究中，我们重新访问了4个突变的寄生虫系，这些寄生虫系是独立选择的，因为它们不能响应离子载体而外出。在所有四个突变体中，我们已经确定钙依赖激酶3作为负责缺陷的基因。我们已经表明，这些突变体中的两个，这是在遗传背景，允许毒力研究，也有一个强大的表型在体内。也就是说，寄生虫不能在小鼠体内形成潜伏期。这项工作提供了重要的信息，即一个单一的激酶是负责形成的潜伏期是重要的寄生虫的传播。
Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle. Toxoplasma gondii, an important human pathogen, is an obligate intracellular parasite, thus getting in and out of cells is key for its survival. The process by which Toxoplasma exits cells, known as egress, is controlled by calcium fluxes and can be triggered by ionophores. In vivo, rapid egress from the host cell has been identified as a means to escape attack by the innate immune system. At the molecular level, calcium dependent events in this parasite are regulated in part by plant like calcium dependent kinases, which share no homology to human kinases and are thus ideal drug targets. In this study we revisited 4 mutant parasite lines that were independently selected for an inability to undergo egress in response to ionophores. In all four mutants we have identified the Calcium Dependent Kinase 3 as the gene responsible for the defects. We have shown that two of these mutants, which are in a genetic background that allows virulence studies, also have a strong phenotype in vivo. That is, the parasites fail to form latent stages in mice. This work provides important information that a single kinase is responsible for the formation of latent stages that are important for transmission of the parasite.
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DOI：
10.1083/jcb.200309097
发表时间：
2004-06-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Arrizabalaga G;Ruiz F;Moreno S;Boothroyd JC
通讯作者：
Boothroyd JC
影响因子：
1.5
作者：
Gubbels, MJ;Wieffer, M;Striepen, B
通讯作者：
Striepen, B
DOI：
10.1073/pnas.97.11.6224
发表时间：
2000-05-23
影响因子：
11.1
作者：
Hwang, I;Sze, H;Harper, JF
通讯作者：
Harper, JF
影响因子：
4
作者：
Arrizabalaga, G;Boothroyd, JC
通讯作者：
Boothroyd, JC
影响因子：
6.4
作者：
Camoni, L;Fullone, MR;Aducci, P
通讯作者：
Aducci, P