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

人类寄生虫溶组织内阿米巴早期成囊基因的鉴定和表征

• 批准号: 10647086
• 负责人: CHERYL Jean INGRAM-SMITH
• 金额: $ 22.42万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-03 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647086
• 关键词: Acetate-CoA Ligase; Acetates; Amebic Liver Abscess; Amebic colitis; Back; Bioinformatics; Candidate Disease Gene; Carrier State; Cell Density; Cell Wall; Cells; Cessation of life; Chitin; Coenzyme A; Cues; Cyst; Data; Developing Countries; Disease; Distant; Entamoeba histolytica; Entamoeba invadens; Environment; Expression Library; Food Contamination; Foundations; Gene Expression Profiling; Gene Library; Gene Silencing; Genes; Genetic; Glucose; Goals; Growth; Heat Stress Disorders; Human; In Vitro; Infection; Ingestion; Investigation; Knowledge; Laboratories; Laboratory culture; Large Intestine; Libraries; Mediating; Methods; Mission; Oxidative Stress; Parasites; Persons; Physiological; Play; Process; Propionates; Protocols documentation; Public Health; Regulation; Regulator Genes; Reproducibility; Reptiles; Research; Role; Sampling; Scientist; Signal Pathway; Signal Transduction; Small Intestines; Stimulus; Stress; System; Technology; Time; Translating; United States National Institutes of Health; Volatile Fatty Acids; biological adaptation to stress; candidate identification; cell motility; contaminated water; deprivation; excystation; human pathogen; interest; nitrosative stress; overexpression; pathogen; prevent; programs; response; screening; thioester; transcription factor; transcriptome sequencing

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.

项目概要 溶组织内阿米巴无法在实验室环境中形成感染性包囊，这极大地阻碍了 对这种人类病原体感染和疾病周期中这一关键阶段的调查 全世界每年约有 1 亿人感染阿米巴痢疾。相反，科学家们被迫依赖 对远亲爬行动物病原体内阿米巴入侵的研究。我们研究的长期目标 计划的目的是确定溶组织内阿米巴如何适应其在感染过程中遇到的不同环境，以及 疾病过程。我们特别感兴趣的是溶组织内阿米巴如何适应大型环境。 肠道，以便在那里定植并通过感染性包囊的形成和传播来传播疾病。我们 现在已经建立了用于培养中溶组织内阿米巴的包囊和脱囊的可重复系统。这 重大技术进步使我们能够了解溶组织内阿米巴如何感知和 对环境信号作出反应，这些信号发出从运动滋养体到传染性包囊并返回的信号转换。作为 作为我们长期目标的一部分，该提案的总体目标是识别和表征基因 负责成囊的启动。拟议项目的基本原理是了解 E. 溶组织细胞通过包囊感知其环境并做出反应，这将有助于更好地理解 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繁衍 它的传染周期。我们将追求两个具体目标：（1）使用 RNAseq 鉴定成囊起始基因； (2)筛选参与溶组织内阿米巴成囊启动的基因的过表达文库。 通过这两种方法鉴定的候选基因将通过基因沉默分析进行验证 和基因过度表达菌株。我们将评估这些菌株的包囊、排出和建立的能力 标准滋养体生长及其对其他应激（如热、氧化和应激）的反应 亚硝化应激以确定是否有任何候选基因发挥一般应激反应作用。作为一部分 在拟议的研究中，我们将优化我们的成囊协议并确定其他环境信号 触发更快的成囊。互补的 RNAseq 和文库筛选方法应该 使我们能够识别几丁质细胞壁形成之前成囊最早阶段所需的基因 作为调节基因。这项研究的意义在于我们现在可以开始了解 调节包囊的环境信号以及溶组织内阿米巴如何作用这些信号。这 研究将对该领域产生重要影响，因为这是第一次涉及阶段的过程 可以直接在人类病原体中充分研究转化，以便更好地了解大肠杆菌如何转化。 溶组织病菌可以在定植期间茁壮成长，并通过传染性包囊的传播继续传播疾病。