Identification and characterization of early encystation genes in the human parasite Entamoeba histolytica

项目总结 溶组织内阿米巴在实验室环境中无法形成感染性包囊，这极大地阻碍了 对这种人类病原体感染和疾病循环中这一关键阶段的调查 全球每年约有1亿人患有阿米巴痢疾。相反，科学家们被迫依赖于 关于爬行动物远亲病原体入侵内阿米巴的研究。我们研究的长期目标是 计划是确定溶解组织杆菌如何适应它在感染和感染期间遇到的不同环境 疾病的过程。我们特别感兴趣的是，溶组织棘球藻是如何适应大的 肠道，以便在那里定居，并通过形成和传播感染性包囊来传播疾病。我们 现已建立了一套可重复使用的溶组织埃希氏菌包囊和胞外培养体系。这 重大的技术进步使我们能够深入了解溶组织肠杆菌是如何感觉和 对环境信号做出反应，这些信号表明从可移动的滋养体转换为感染性包囊，然后再转换回来。AS 作为我们长期目标的一部分，这项提案的总体目标是识别和表征基因 负责包衣的启动。提出这个项目的理由是，理解E。 溶解组织通过包裹来感知和响应环境将有助于更好地理解 当这种病原体在不同阶段遇到非常不同的环境时如何生存和茁壮成长 它的传染周期。我们将追求两个特定的目标：(1)使用RNAseq鉴定囊化起始基因； 以及(2)筛选溶组织乳杆菌囊化起始相关基因的过表达文库。 通过这两种方法确定的候选基因将通过对沉默基因的分析来验证 和基因过表达菌株。我们将评估这些菌株的包囊、包囊和建立 滋养体的标准生长以及它们对其他胁迫的反应，如热、氧化和 亚硝化性应激，以确定是否有任何候选基因发挥一般应激反应的作用。作为一部分 在拟议的研究中，我们将优化我们的加密方案，并确定其他环境信号 这会引发更快的堆积。互补的RNAseq和文库筛选方法应该 使我们能够确定甲壳素细胞壁形成之前囊化的最早阶段所需的基因 作为调控基因。这项研究的意义在于，我们现在可以开始了解 调节囊化作用的环境信号，以及这些信号是如何被溶组性肠杆菌作用的。这 研究将对该领域产生重要影响，因为这是第一次涉及到阶段的过程 可以直接在人类病原体中进行充分的转换研究，以更好地了解E. 在定植期间，溶组织线虫可以蓬勃发展，并通过传染性包囊的传播继续传播疾病。

PROJECT SUMMARY The inability of Entamoeba histolytica to form infectious cysts in the laboratory setting has greatly hindered investigation of this crucial stage in the infection and disease cycle of this human pathogen that causes amoebic dysentery in ~100 million people each year worldwide. Instead, scientists have been forced to rely on studies with the distantly related reptile pathogen Entamoeba invadens. The long-term goal of our research program is to determine how E. histolytica adapts to different environments it encounters during infection and the disease process. In particular, we are interested in how E. histolytica adapts to the environment of the large intestine in order to colonize there and spread disease by formation and dissemination of infectious cysts. We have now established a reproducible system for encystation and excystation of E. histolytica in culture. This major technological advance enables us to pursue an understanding of how E. histolytica senses and responds to environmental cues that signal conversion from motile trophozoite to infectious cyst and back. As part of our long-term goal, the overall objective of this proposal is to identify and characterize genes responsible for initiation of encystation. The rationale for the proposed project is that understanding how E. histolytica senses and responds to its environment through encystation will lead to a better understanding of how this pathogen can survive and thrive as it encounters very diverse environments during different stages of its infectious cycle. We will pursue two specific aims: (1) identify encystation initiation genes using RNAseq; and (2) screen an overexpression library for genes involved in initiation of encystation in E. histolytica. Candidate genes identified through these two approaches will be validated through analysis of gene silenced and gene overexpression strains. We will evaluate these strains for their ability to encyst, excyst, and establish standard trophozoite growth as well as their responses to other stresses such as heat, oxidative, and nitrosative stress to determine whether any of the candidate genes play a general stress response role. As part of the proposed research, we will optimize our encystation protocol and determine other environmental signals that trigger more rapid encystation. The complementary RNAseq and library screening approaches should allow us to identify genes required for the earliest stages of encystation prior to chitin cell wall formation as well as regulatory genes. The significance of this research is that we can now begin to understand the interplay of environmental signals that regulate encystation and how these signals are acted upon by E. histolytica. This research will have an important impact on the field in that for the first time the processes involved in stage conversion can be fully studied directly in the human pathogen to provide a better understanding of how E. histolytica can thrive during colonization and continue to propagate disease through spread of infectious cysts.