Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay.
Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay.
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DOI：
10.3791/60985
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发表时间：
2020-04-29
期刊：
影响因子：
--
通讯作者：
Dou Z
中科院分区：
文献类型：
--
作者：
Key M;Bergmann A;Micchelli C;Thornton LB;Millard S;Dou Z
弓形虫是一种广泛影响人类的原生动物病原体。目前用于临床治疗弓形虫病的抗生素有限。此外，它们在某些人群中表现出不良副作用。因此，临床弓形虫病的新疗法的发现势在必行。新型抗生素开发的第一步是使用高通量筛选策略鉴定在抑制寄生虫生长方面显示出高功效的化合物。作为一种专性细胞内病原体，弓形虫只能在宿主细胞内复制，这禁止使用吸光度测量作为生长的快速指标。这里提供的是一个详细的协议为基础的生长试验。例如，该方法用于计算野生型弓形虫寄生虫的倍增时间，并测量吗啉脲-亮氨酰-高苯基-乙烯基砜苯基（LHVS，半胱氨酸蛋白酶靶向化合物）抑制寄生虫细胞内生长的功效。还描述了在弓形虫中使用50 bp同源区进行同源依赖性重组（HDR）的基于CRISPR-Cas9的基因缺失方案。通过定量LHVS在野生型和TgCPL（弓形虫组织蛋白酶L样蛋白酶）缺陷型寄生虫中的抑制功效，表明LHVS比Δcpl生长更有效地抑制野生型寄生虫生长，表明TgCPL是LHVS在弓形虫中结合的靶标。该方法灵敏度高、操作简便，适用于监测弓形虫增殖和高通量评价药物疗效。
Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than Δcpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.
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影响因子：
3.7
作者：
Sidik SM;Hackett CG;Tran F;Westwood NJ;Lourido S
通讯作者：
Lourido S
DOI：
10.1111/j.1550-7408.1977.tb04774.x
发表时间：
1977-01-01
期刊：
JOURNAL OF PROTOZOOLOGY
影响因子：
--
作者：
PFEFFERKORN, ER;PFEFFERKORN, LC
通讯作者：
PFEFFERKORN, LC
影响因子：
2.4
作者：
Lindsay, D S;Dubey, J P
通讯作者：
Dubey, J P
影响因子：
6.4
作者：
Dou Z;McGovern OL;Di Cristina M;Carruthers VB
通讯作者：
Carruthers VB
CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.