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.

项目摘要 溶组织内阿米巴在实验室环境中无法形成传染性囊肿，这极大地阻碍了 调查这一关键阶段的感染和疾病周期的这种人类病原体， 全世界每年约有1亿人感染阿米巴痢疾。相反，科学家们被迫依赖于 与爬行动物病原体入侵内阿米巴关系较远的研究。我们研究的长期目标是 程序是确定如何E.溶组织菌适应感染过程中遇到的不同环境， 疾病的过程。特别是，我们感兴趣的是如何E。组织溶解菌适应大环境 肠道以便在那里定居并通过传染性囊肿的形成和传播来传播疾病。我们 建立了一个可重复的E.溶组织细胞。这 一项重大的技术进步使我们能够理解E.溶组织感觉， 对环境信号作出反应，信号从活动的滋养体转化为传染性包囊，然后再转化回来。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 负责包囊形成的起始。该项目的基本原理是理解E. 溶组织菌通过成囊来感知和响应其环境，这将有助于更好地理解 这种病原体如何在不同阶段遇到非常不同的环境时生存和繁殖， 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; 和（2）筛选参与E.溶组织剂 通过这两种方法鉴定的候选基因将通过分析沉默的基因进行验证。 和基因过表达菌株。我们将评估这些菌株的成囊、出囊和建立 标准滋养体生长以及它们对其它应激如热、氧化和 亚硝化胁迫，以确定是否有任何候选基因发挥一般的应激反应的作用。一部分 在拟议的研究中，我们将优化我们的包囊方案，并确定其他环境信号 从而引发更快的包囊形成互补的RNAseq和文库筛选方法应 也使我们能够鉴定出在几丁质细胞壁形成之前包囊形成的最早阶段所需的基因 as regulatory调控genes基因.这项研究的重要性在于，我们现在可以开始了解 环境信号调节成囊以及这些信号如何被E.溶组织剂这 研究将对该领域产生重要影响，因为第一次涉及阶段的过程 转换可以直接在人类病原体中进行充分研究，以更好地了解E. 溶组织菌可在定殖期间繁殖并通过传染性包囊的传播继续传播疾病。