A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment.
A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment.
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DOI：
10.1128/msphere.00769-20
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发表时间：
2020-10-07
期刊：
影响因子：
4.8
通讯作者：
Hovel-Miner G
中科院分区：
文献类型：
--
作者：
Carter M;Gomez S;Gritz S;Larson S;Silva-Herzog E;Kim HS;Schulz D;Hovel-Miner G
锥虫寄生虫威胁着全球超过10亿人的健康。由于它们的基因组与那些已经建立的真核生物的基因组高度分化，保守性在分配基因功能时并不总是有用的。然而，正是在锥虫特异性基因中才有可能找到理想的治疗靶点，正向遗传学方法是发现新基因功能的有效途径。我们使用ORFeome方法克隆了大比例的布氏锥虫基因，并产生了功能获得性寄生虫文库。该文库被用于遗传筛选，以确定促进对临床上重要但毒性很大的药物美拉胂醇耐药性的基因。从屏幕上产生的命中证明了库的有用性，在确定已知的途径，并发现新的方面的电阻介导的蛋白质定位到鞭毛和鞭毛。本文产生的强大的新的遗传工具，预计将促进锥虫生物学和治疗发展的进步在未来几年。布氏锥虫是一种早期分支的原生动物寄生虫，引起人类和动物非洲锥虫病。正向遗传学方法是揭示锥虫生物学、发病机制和治疗方法的新方面的有力工具。在这里，我们生成了一个T。Brucei克隆了由靶向的7，245个基因的>90%组成的ORFeome，并使用它来制备广泛适用于大规模正向遗传筛选的诱导型功能获得性寄生虫文库。我们进行了一项原理验证遗传筛选，以确定其表达促进美拉胂醇（一种关键的最后药物）生存的基因。在美拉胂醇幸存者群体中被确定为过度表达的57个基因包括编码已确定药物靶标（锥虫硫酮）生物合成限速酶的基因，从而验证了该工具。此外，还鉴定了与基因表达、鞭毛定位和鞭毛体定位相关的新基因，这些基因的一个子集在培养物中过表达后增加了美拉胂醇耐药性。这些发现提供了新的见解锥虫的基础生物学，药物靶点的影响，直接或间接的耐药机制。这项研究产生了一个T。布氏杆菌ORFeome和功能获得性寄生虫文库，证明了该文库在正向遗传筛选中的有用性，并确定了美拉胂醇耐药性的新方面，这将是未来研究的主题。这些强大的遗传工具可用于广泛推进锥虫研究。锥虫寄生虫威胁着全球超过10亿人的健康。由于它们的基因组与那些已经建立的真核生物的基因组高度分化，保守性在分配基因功能时并不总是有用的。然而，正是在锥虫特异性基因中才有可能找到理想的治疗靶点，正向遗传学方法是发现新基因功能的有效途径。我们使用ORFeome方法克隆了大比例的布氏锥虫基因，并产生了功能获得性寄生虫文库。该文库被用于遗传筛选，以确定促进对临床上重要但毒性很大的药物美拉胂醇耐药性的基因。从屏幕上产生的命中证明了库的有用性，在确定已知的途径，并发现新的方面的电阻介导的蛋白质定位到鞭毛和鞭毛。本文产生的强大的新的遗传工具，预计将促进锥虫生物学和治疗发展的进步在未来几年。
Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library’s usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come. Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library’s usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research. IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library’s usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.