Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection
Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection
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全基因组 CRISPR 筛选鉴定弓形虫感染所需的宿主因子
DOI：
10.3389/fcimb.2019.00460
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发表时间：
2020-01-22
影响因子：
5.7
通讯作者：
Peng, Hong-Juan
中科院分区：
文献类型：
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作者：
Wu, Shui-Zhen;Wei, Hai-Xia;Peng, Hong-Juan
刚地弓形虫是专性细胞内原生动物，由于其基因组较小且编码的蛋白质有限，它们必须利用宿主因子来入侵、复制和传播。这类宿主因子可被定义为宿主依赖因子（HDFs）。尽管宿主依赖因子对细胞活力并非必需，但它们对病原体感染至关重要，是治疗干预的潜在理想靶点。然而，关于刚地弓形虫感染所需的这些宿主依赖因子的信息非常匮乏。在本研究中，利用慢病毒CRISPR - Cas9 - sgRNA文库对人包皮成纤维细胞（HFF）的基因进行了全面编辑，然后用感染复数为1（MOI = 1）的刚地弓形虫感染慢病毒处理过的细胞10天，以鉴定对刚地弓形虫感染至关重要的宿主依赖因子。收集存活细胞并送去进行sgRNA测序。与sgRNA序列匹配的基因或微小RNA（miRNAs）是潜在的宿主依赖因子。在刚地弓形虫感染后，慢病毒处理组中的一些细胞比未处理对照组中的细胞存活时间更长。从19050个人类基因和1864个人类初级微小RNA中，鉴定出1193个潜在的宿主依赖因子，包括1183个基因和10个初级微小RNA（对应17个成熟微小RNA）。其中，7个基因和5个成熟微小RNA分别通过小干扰RNA（siRNAs）、微小RNA抑制剂和模拟物得到了验证。生物信息学分析显示，在1183个基因中，53个潜在的宿主依赖因子与宿主肌动蛋白细胞骨架的调节有关，23个潜在的宿主依赖因子编码免疫负调控因子。这一结果表明，肌动蛋白动力学对刚地弓形虫感染不可或缺，一些宿主免疫负调控因子可能参与解除宿主防御。我们的研究结果增加了目前对刚地弓形虫感染所需宿主因子的有限认识，并为药物治疗和疫苗开发提供了新的靶点。
Toxoplasma gondii are obligate intracellular protoza, and due to their small genome and limited encoded proteins, they have to exploit host factors for entry, replication, and dissemination. Such host factors can be defined as host dependency factors (HDFs). Though HDFs are inessential for cell viability, they are critical for pathogen infection, and potential ideal targets for therapeutic intervention. However, information about these HDFs required by T. gondii infection is highly deficient. In this study, the genes of human foreskin fibroblast (HFF) cells were comprehensively edited using the lentiviral CRISPR-Cas9-sgRNA library, and then the lentivirus-treated cells were infected with T. gondii at multiplication of infection 1 (MOI = 1) for 10 days to identify HDFs essential for T. gondii infection. The survival cells were harvested and sent for sgRNA sequencing. The sgRNA sequence matched genes or miRNAs were potential HDFs. Some cells in the lentivirus-treated group could survive longer than those in the untreated control group after T. gondii infection. From a pool of 19,050 human genes and 1,864 human pri-miRNAs, 1,193 potential HDFs were identified, including 1,183 genes and 10 pri-miRNAs (corresponding with 17 mature miRNAs). Among them, seven genes and five mature miRNAs were validated with siRNAs, miRNA inhibitors, and mimics, respectively. Bioinformatics analysis revealed that, among the 1,183 genes, 53 potential HDFs were associated with regulation of host actin cytoskeleton and 23 potential HDFs coded immune negative regulators. This result indicated that actin dynamics were indispensable for T. gondii infection, and some host immune negative regulators may be involved in disarming host defenses. Our findings contribute to the current limited knowledge about host factors required by T. gondii infection and provide us with new targets for medication therapy and vaccine exploitation.