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.

概括动力质体寄生虫是单细胞的真核寄生虫，其中一些是原因毁灭性的人类疾病，包括查加斯病，利什曼病和人类非洲人锥虫病。致病动力质体是由昆虫向量传播的。这些寄生虫载体关系是特异性的，具有不同种类的寄生虫物种。而它们各自的昆虫宿主中的动质寄生虫的生命周期可能有所不同，一个共同的特征是寄生虫遵守昆虫组织。粘合剂阶段对于定植昆虫，在某些情况下允许发展感染形式。对于所有动力质体的粘附本身具有共享的超微结构特征，并且类似于Hemidesmosmos。分子该粘合剂结构的组成部分以及触发其形成的信号通路是完全未知。 Crithidia fasciculata是一种仅感染一个宿主蚊子的寄生虫。这是通常不认为是人类病原体；但是，有人类的报道感染，通常在免疫功能低下的患者或与利什曼原虫属的共同感染中。 C. fasciculata多年来一直被用作探索动力质体基本生物学的模型寄生虫。它们代表了调查粘附机制的强大系统，因为它们将不仅遵守其蚊子宿主的后肠，还遵守人造底物（例如组织培养物）塑料。这使我们能够使用体外测定来确定各种候选蛋白和粘附过程中的途径。此外，我们可以观察到粘附过程的阶段实时使用活细胞成像。我们假设寄生虫的粘附阶段是一种独特的发展形式，并且与该形式的区分是由特定的信号转导途径介导的。此外，我们预测这种粘附是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特定目的：（1）确定环状AMP信号通路的作用调节粘附，（2）建立了快速创造遗传敲除的条件。使用CRISPR/CAS9的fasciculata。该项目建立在我们使用RNASEQ进行比较的我们发表的工作的基础上粘附和游泳细胞的基因表达谱，并将为高通量设定阶段确定大量候选蛋白在体外粘附中的作用的方法，这可以然后在体内评估其定植蚊子的能力。拟议工作的结果将改进用于遗传操纵fasciculata的工具，该工具将使研究人员使用该模型，以及对各种动力质体物种粘附的共享机制的见解昆虫宿主。