Large-scale CRISPR screening of essential calcium-related genes in the human parasite Tr. cruzi

摘要 查加斯病（CD）是一种由原生动物寄生虫克氏锥虫引起的寄生性热带疾病。它 全世界有600万至700万人感染，没有疫苗或令人满意的治疗方法。历史上， CD被认为是拉丁美洲的地方病，但最近它已蔓延到欧洲，北美， 以及太平洋地区，因为全球移民的缘故。据估计，美国目前有30万人感染了艾滋病毒。 美国，其中45,000人表现出心肌病，而大多数病例仍然未被诊断和治疗。那里 只有两种被接受的治疗恰加斯病的药物，但它们只能通过中心获得， 疾病控制和预防（CDC）。除了它们的副作用，这些药物的疗效 一个人感染的时间越长，因此，开发新的无毒 治疗慢性恰加斯病患者的化学疗法是高度需要的。 我们感兴趣的是研究可用作化疗靶点的蛋白质， 有效治疗恰加斯病。我们的方法是确定代谢途径，缺乏在 人类细胞，但必须寄生虫生存。钙离子（Ca ~（2+））是细胞内重要的第二信使， 调节大量细胞过程的锥虫。于T. cruzi，Ca 2+信号尤其是 对于宿主细胞侵袭、分化、细胞增殖调节和细胞生物能量学是重要的。几种蛋白质 在锥虫中已经鉴定出参与钙稳态。然而，T. cruzi基因组含有 各种各样的钙相互作用的未知蛋白质。使用CRISPR/Cas9系统，我们已经 能够进行T的敲除、互补和原位标记。克鲁兹基因。此外，我们有 在T. cruzi及其同系物 哺乳动物细胞基于CRISPR的诱导沉默系统的开发在T.克鲁兹和 我们将用它来进行一个大规模的筛选基因编码的钙相互作用蛋白质在这个 有机体然后，我们将确定参与钙信号传导的必要蛋白质，并进行其生物学行为。 作为药物靶点的验证，以及其他已经鉴定的必需蛋白质。我们终于可以公开 有关蛋白质功能、相互作用伴侣、细胞定位甚至抑制剂的相关信息。 必需的钙相关蛋白质。总而言之，这些结果最终将在化学物质中达到顶峰 验证它们作为治疗南美锥虫病的候选靶点。 该项目的指导阶段将在Roberto Docampo博士的实验室进行， 格鲁吉亚大学，隶属于热带和新兴全球疾病中心。的 学术环境，基础设施和实验室动态整合完美的条件，以完成我的 培训计划，并将是决定性的，以实现我的实际目标：成为一个独立的研究人员和 领导科学家，为生物医学领域知识的产生做出贡献。

Abstract Chagas disease (CD) is a parasitic tropical pathology caused by the protozoan parasite Trypanosoma cruzi. It affects 6-7 million people worldwide, for which no vaccine or satisfactory treatment are available. Historically, CD has been considered endemic in Latin America, but more recently it has spread to Europe, North America, and the Pacific, due to global migrations. An estimated 300,000 people are currently infected in the United States, 45,000 of which exhibit cardiomyopathy, while most cases remain undiagnosed and untreated. There are only two accepted drugs to treat Chagas disease, but they are only available through the Centers for Disease Control and Prevention (CDC). Besides their adverse side effects, the efficacy of these drugs decreases the longer a person has been infected. Therefore, the development of new non-toxic chemotherapies to treat chronic patients of Chagas disease is highly demanded. We are interested in investigating proteins that can be used as chemotherapeutic targets for developing effective treatments against Chagas disease. Our approach is to identify metabolic pathways that are absent in human cells but necessary for parasite survival. Calcium ion (Ca2+) is an important second messenger in trypanosomatids that regulates a vast repertoire of cellular processes. In T. cruzi, Ca2+ signaling is particularly important for host cell invasion, differentiation, osmoregulation and cell bioenergetics. Several proteins involved in calcium homeostasis have been identified in trypanosomes. However, the T. cruzi genome contains a wide variety of calcium-interacting uncharacterized proteins. Using the CRISPR/Cas9 system, we have been able to perform knockout, complementation and in situ tagging of T. cruzi genes. Furthermore, we have established important differences between calcium signaling proteins in T. cruzi and their homologs in mammalian cells. The development of a CRISPR-based inducible silencing system is feasible in T. cruzi and we will use it to perform a large-scale screening of genes encoding calcium-interacting proteins in this organism. Then we will identify essential proteins involved in calcium signaling and perform their biological validation as drug targets, together with other essential proteins already identified. We finally expect to disclose relevant information on protein function, interacting partners, cellular localization and even inhibitors of essential calcium-related proteins. Taken together these results will eventually culminate in the chemical validation of them as target candidates for treatment development against Chagas disease. The mentored phase of this project is going to be developed in the laboratory of Dr. Roberto Docampo at the University of Georgia, which belongs to the Center for Tropical and Emerging Global Diseases. The academic environment, infrastructure and laboratory dynamics integrate the perfect conditions to complete my training plan, and will be determinant to achieve my actual goal: to become an independent researcher and a leader scientist, contributing to the generation of knowledge in the biomedical field.