The role of stem-cell mediated midgut repair in the dynamics of mosquito infections

干细胞介导的中肠修复在蚊子感染动态中的作用

• 批准号: 10404118
• 负责人: Nicolas Buchon
• 金额: $ 51.04万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-05 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10404118
• 关键词: ATAC-seq; Acute; Address; Aedes; Affect; Age; Animal Model; Animals; Antimitotic Agents; Area; Bacteria; Biology; Blood; Cell Differentiation process; Cells; Cessation of life; Communicable Diseases; Communities; Competence; Complex; Culicidae; Disease; Drosophila genus; Enterocytes; Enteroendocrine Cell; Epithelial; Epithelial Cells; Epithelial Physiology; Equilibrium; Fluorescence-Activated Cell Sorting; Genetic; Genetic Transcription; Goals; Homeostasis; Human; Infection; Intestines; Investigation; Knowledge; Longevity; Malaria; Mediating; Midgut; Modeling; Natural regeneration; Oral; Oral Ingestion; Outcome; Pathogenicity; Pathway interactions; Phenotype; Physiological; Physiology; Play; Population; Regenerative response; Reporting; Research; Resolution; Role; Stimulus; Stress; Testing; Tissues; Transgenic Organisms; Vector-transmitted infectious disease; Vectorial capacity; Virus; Work; cell type; epithelial repair; feeding; gastrointestinal epithelium; human pathogen; innovation; microbial; mosquito-borne; novel; oral infection; oral pathogen; pathogen; progenitor; repaired; response; single-cell RNA sequencing; stem cell proliferation; stem cells; stem-like cell; success; tool; transcriptome sequencing; transmission process; vector; vector control; vector mosquito; virtual

Project Summary Vector-borne diseases account for more than 17% of all infectious disease and cause more than 700,000 deaths annually 1. Mosquitoes alone cause 400,000 malaria deaths and transmit viruses to hundreds of millions 2. The vectorial capacity of mosquitoes depends on their ability to survive infection. The damaging effects of pathogenic invasion of the mosquito midgut are well-documented 15-22, but little is known about how mosquitoes tolerate this stress. Intestinal stem cell (ISC) mediated midgut epithelial repair is essential for Drosophila survival following oral ingestion of pathogens 33. The mosquito midgut epithelium contains ISC-like cells 27, 34, 37-39, but their functional significance for infection outcomes and mosquito survival is unknown. We propose to address this knowledge gap in vector biology by investigating the mosquito gut regenerative response to pathogenic invasion. The “black box” regarding the functional significance of ISCs in the mosquito midgut is part of a fundamental knowledge gap: physiological studies treat the mosquito midgut as a homogeneous whole, rather than a complex, regionally compartmentalized tissue comprised of multiple cell populations (e.g. enterocytes, enteroendocrine cells, and ISCs). The specific contributions of these cell types to gut-pathogen interactions have not been investigated. The proposed work will not only illuminate mosquito epithelial responses to infection at the cellular level but will lead to the creation of new and innovative tools for the broader vector biology community. The first two aims of this project are (A) to characterize gut epithelial cell dynamics in mosquitoes under conditions of homeostasis and oral infection and (B) to evaluate the role of midgut epithelial repair in mosquito infection outcomes. Aedes aegypti will be used as a model to determine what stimuli (including human pathogens) affect gut epithelial turnover rates, whether post-infection repair rebuilds the gut homeostatically or alters epithelial composition, what genetic pathways control midgut epithelial repair, and what role epithelial repair plays in vector survival and competence. Our third aim is (C) to determine the specific contributions of functionally differentiated cell populations to epithelial dynamics and infection response. We will use single-cell RNAseq/ATACseq to establish how many cell types compose the midgut epithelium, create new transgenic lines expressing fluorescent markers for important cell types (enteroendocrine cells and ISCs), and, using these lines, couple fluorescence-activated cell sorting with RNAseq to examine the transcriptional response of the three major cell types (enterocytes, enteroendocrine cells, and ISCs) to infection. Our study will fill a critical gap in our understanding of mosquito midgut regenerative responses to pathogenic invasion. Our long-term goal is to identify new targets for vector control strategies that disrupt gut regeneration and reduce survival of infected mosquitoes below the critical incubation threshold required for pathogen transmission. In addition to laying the groundwork for innovative control targets, we will create tools for the broader vector biology community, paving the way for novel discoveries in mosquito midgut physiology.

项目摘要 媒介传播疾病占所有传染病的17%以上，造成70多万人死亡 每年1.仅蚊子就导致40万人死于疟疾，并将病毒传播给数亿人2. 蚊子的媒介能力取决于它们在感染后存活的能力。致病因素的危害作用 蚊子中肠的入侵有很好的记录15-22，但关于蚊子如何耐受这一点知之甚少 压力。肠干细胞(ISC)介导的中肠上皮修复对果蝇存活至关重要 口服摄入病原体33.蚊子中肠上皮含有ISC样细胞27、34、37-39，但它们的功能 对感染结果和蚊子存活的意义尚不清楚。我们建议解决这一知识 通过研究蚊子肠道对病原体入侵的再生反应来研究媒介生物学方面的差距。 关于蚊子中肠中ISCs功能重要性的“黑匣子”是一个基本的 知识鸿沟：生理学研究将蚊子中肠视为一个同质的整体，而不是 由多个细胞群组成的复杂的、区域分隔的组织(例如，肠细胞， 肠内分泌细胞和ISCs)。这些细胞类型对肠道-病原体相互作用的特殊贡献有 没有被调查过。拟议的工作不仅将阐明蚊子上皮细胞对感染的反应 这不仅限于细胞层面，而且将导致为更广泛的媒介生物界创造新的创新工具。 该项目的前两个目标是(A)描述蚊子肠道上皮细胞的动力学。 动态平衡和口腔感染的条件和(B)评估中肠上皮修复在口腔感染中的作用 蚊子感染的后果。埃及伊蚊将被用作模型，以确定什么刺激(包括 人类病原体)影响肠道上皮周转率，感染后修复是否重建肠道 动态平衡或改变上皮成分，什么遗传途径控制中肠上皮修复，以及什么 上皮修复在病媒生存和能力中发挥作用。我们的第三个目标是(C)确定具体的 功能分化细胞群对上皮细胞动力学和感染的贡献 回应。我们将使用单细胞RNAseq/ATACseq来确定组成中肠的细胞类型 上皮细胞，创建新的转基因株系，表达重要细胞类型(肠道内分泌)的荧光标记 细胞和间充质干细胞)，并使用这些系，将荧光激活的细胞分类与RNAseq结合起来，以检查 三种主要细胞类型(肠细胞、肠内分泌细胞和ISCs)对感染的转录反应。 我们的研究将填补我们在理解蚊子中肠对致病菌的再生反应方面的一个关键空白 入侵。我们的长期目标是为干扰肠道再生的病媒控制策略确定新的目标 并将受感染蚊子的存活率降低到病原体所需的临界孵化阈值以下 变速箱。除了为创新的控制目标奠定基础外，我们还将为 更广泛的媒介生物学社区，为蚊子中肠生理学的新发现铺平了道路。