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Functional analysis of insect-specific adhesion in a model kinetoplastid

模型动质体中昆虫特异性粘附的功能分析

基本信息

批准号：
10170257
负责人：
Megan Povelones
金额：
$ 20.21万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10170257
关键词：
Address Adherence Adhesions Adhesives African Trypanosomiasis Arthropods Biological Assay Biology Brazil CRISPR/Cas technology Candidate Disease Gene Cell Line Cells Chagas Disease Characteristics Chemicals Complex Crithidia Crithidia fasciculata Culicidae Cyclic AMP DNA Structure Development Disease Eukaryotic Cell Family Follow-Up Studies Gene Expression Gene Expression Profiling Generations Genetic Genome Hemidesmosomes Heterogeneity Hindgut Human Immunocompetent Immunocompromised Host In Vitro Infection Insect Vectors Insecta Knock-out Laboratory Infection Leishmania Leishmaniasis Leptomonas Life Cycle Stages Mediating Metabolism Mitochondrial DNA Modeling Molecular Morphology Nutrient Outcome Parasites Pathogenicity Pathway interactions Patients Phenotype Phylogenetic Analysis Process Production Proteins Publishing Regulation Reporting Research Personnel Resistance Resources Role Signal Pathway Signal Transduction Signal Transduction Pathway Structure Swimming System Testing Tissues Transcript Trypanosoma brucei brucei Trypanosoma cruzi Visceral Leishmaniasis Work adhesion process burden of illness co-infection emerging pathogen genetic manipulation high throughput screening high throughput technology human disease human pathogen improved in vitro Assay in vivo insight live cell imaging live cell microscopy novel novel strategies pathogen standard of care technology development therapeutic target time use tissue culture tool transcriptome sequencing transcriptomics transmission process vector virtual

项目摘要

SUMMARY Kinetoplastid parasites are single-celled eukaryotic parasites, some of which are causative agents of devastating human diseases, including Chagas disease, Leishmaniasis, and human African trypanosomiasis. Pathogenic kinetoplastids are transmitted by insect vectors. These parasite-vector relationships are specific, with different insect species harboring different species of parasite. While the life cycles of kinetoplastid parasites in their respective insect hosts can differ, one shared feature is adherence of the parasite to insect tissue. The adhesive stage is necessary for colonization of the insect, and in some cases allows for development of infectious forms. For all kinetoplastids, the adhesion itself has shared ultrastructural features, and resembles a hemidesmosome. The molecular components of this adhesive structure, and the signaling pathways that trigger its formation, are completely unknown. Crithidia fasciculata is a parasite that only infects one host, the mosquito. It is generally not considered to be a human pathogen; however, there have been reports of human infections, typically in immunocompromised patients or in co-infections with Leishmania spp. C. fasciculata has for years been used as a model for exploring the basic biology of kinetoplastid parasites. They represent a powerful system to investigate mechanisms of adhesion since they will adhere not only to the hindgut of their mosquito host, but to artificial substrates such as tissue culture plastic. This allows us to use in vitro assays to determine the role of various candidate proteins and pathways in the process of adhesion. In addition, we can observe the stages of the adhesion process in real time using live-cell imaging. We hypothesize that the adhesive stage of the parasite is a distinct developmental form, and that differentiation to this form is mediated by specific signal transduction pathways. In addition, we predict that adhesion is a multi-stage process involving novel proteins. We will address these hypotheses through the following Specific Aims: (1) Determine the role of the cyclic AMP signaling pathway in regulating adhesion, and (2) Establish conditions for rapid creation of genetic knock-outs in C. fasciculata using CRISPR/Cas9. This project builds upon our published work using RNAseq to compare gene expression profiles of adherent and swimming cells, and will set the stage for a high-throughput approach to determine the role of a large number of candidate proteins in adhesion in vitro, which can then be evaluated in vivo for their ability to colonize mosquitoes. The outcomes of the proposed work will be improved tools for genetic manipulation of C. fasciculata, which will benefit researchers using this model, and insight into shared mechanisms for adhesion of diverse kinetoplastid species to their insect hosts.

概括动质体寄生虫是单细胞真核寄生虫，其中一些是病原体毁灭性的人类疾病，包括南美锥虫病、利什曼病和非洲人类疾病锥虫病。致病动质体通过昆虫媒介传播。这些寄生虫载体关系是特定的，不同的昆虫种类含有不同种类的寄生虫。虽然动质体寄生虫在各自昆虫宿主中的生命周期可能不同，一个共同的特征是寄生虫对昆虫组织的粘附。粘附阶段对于定植是必要的昆虫，并且在某些情况下允许发展感染形式。对于所有动质体，粘附本身具有共同的超微结构特征，并且类似于半桥粒。分子这种粘合结构的组成部分以及触发其形成的信号通路是完全未知。束状短膜虫是一种只感染一个宿主——蚊子的寄生虫。这是通常不被认为是人类病原体；然而，有报道称，人类感染，通常发生在免疫功能低下的患者或利什曼原虫合并感染中。 C. fasciculata 多年来一直被用作探索动质体基础生物学的模型寄生虫。它们代表了研究粘附机制的强大系统，因为它们将不仅粘附在蚊子宿主的后肠上，而且粘附在组织培养物等人造基质上塑料。这使我们能够使用体外测定来确定各种候选蛋白质的作用和粘附过程中的途径。此外，我们可以观察粘合过程的各个阶段使用活细胞成像实时进行。我们假设寄生虫的粘附阶段是一种独特的发育形式，并且向这种形式的分化是由特定的信号转导途径介导的。此外，我们预测粘附是一个涉及新蛋白质的多阶段过程。我们将解决这些假设通过以下具体目标：（1）确定环AMP信号通路在调节粘附，以及（2）为快速产生C. 基因敲除创造条件。使用 CRISPR/Cas9 的束状藻。该项目建立在我们已发表的使用 RNAseq 进行比较的工作的基础上贴壁细胞和游动细胞的基因表达谱，并将为高通量奠定基础确定大量候选蛋白在体外粘附中的作用的方法，可以然后在体内评估它们在蚊子中定殖的能力。拟议工作的成果将成为 C. fasciculata 基因操作的改进工具，这将有利于研究人员使用该模型，以及对不同动质体物种粘附到它们的共享机制的深入了解昆虫宿主。