Functional analysis of insect-specific adhesion in a model kinetoplastid

总结 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，包括恰加斯病、利什曼病和非洲人 锥虫病原动质体由昆虫媒介传播。这些寄生载体 关系是特定的，不同的昆虫种类携带不同种类的寄生虫。而 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。粘附阶段是必要的殖民化的 昆虫，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 这种粘附结构的组成部分，以及触发其形成的信号通路， 完全未知束状短翅虫是一种寄生虫，只感染一种宿主，蚊子。是 一般不认为是人类病原体;然而，有报告称，人类 感染，通常在免疫功能低下的患者中或在与利什曼原虫属的共感染中。 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 不仅附着在蚊子宿主的后肠上， 塑料这使我们能够使用体外测定来确定各种候选蛋白的作用， 粘附过程中的路径。此外，我们还可以观察粘附过程的各个阶段， 真实的时间使用活细胞成像。 我们假设寄生虫的粘附阶段是一种独特的发育形式， 向这种形式的分化由特定的信号转导途径介导。此外，我们预测 粘附是一个涉及新蛋白质的多阶段过程。我们将讨论这些假设 通过以下具体目的：（1）确定环AMP信号通路在 调节粘附，和（2）建立快速创建C. fasciculata使用CRISPR/Cas9.该项目建立在我们发表的工作基础上，使用RNAseq比较 贴壁和游泳细胞的基因表达谱，并将为高通量 方法来确定大量的候选蛋白在体外粘附中的作用，这可以 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 将是C. fasciculata，这将有利于研究人员使用 这一模型，并深入了解不同动质体物种粘附到其 昆虫宿主

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.