Functional analysis of insect-specific adhesion in a model kinetoplastid

摘要 动体寄生虫是一种单细胞真核寄生虫，其中一些是引起 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。致病动体是通过昆虫媒介传播的。这些寄生虫媒介 关系是特定的，不同种类的昆虫携带不同种类的寄生虫。而当 动体寄生虫在它们各自的昆虫宿主中的生命周期可能不同，一个共同的特征是 寄生虫附着在昆虫组织上。黏附阶段是殖民所必需的。 昆虫，在某些情况下允许发展感染形式。对于所有动质体而言， 黏附本身具有相同的超微结构特征，类似于半桥粒。分子 这种黏附结构的组件以及触发其形成的信号通路是 完全未知。束毛虫是一种寄生虫，只感染一种宿主--蚊子。它是 一般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与利什曼原虫混合感染的患者中。 多年来，束毛藻一直被用作探索动叶绿体基本生物学的模型。 寄生虫。它们代表了一个强大的系统来研究粘着机制，因为它们将 不仅附着在蚊子宿主的后肠上，还附着在人工基质上，如组织培养 塑料的。这使得我们可以使用体外分析来确定各种候选蛋白质和 黏附过程中的路径。此外，我们还可以观察到粘连过程的各个阶段 使用活细胞成像技术进行实时检测。 我们假设寄生虫的粘附期是一种不同的发育形式，并且 分化为这种形式是通过特定的信号转导途径。此外，我们预测 这种黏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)确定环状AMP信号通路在 调节粘附性；(2)创造快速产生遗传敲除的条件。 利用CRISPR/Cas9.该项目建立在我们发布的工作基础上，使用RNAseq进行比较 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 一种确定大量候选蛋白在体外黏附中作用的方法，可以 然后在体内对它们定植蚊子的能力进行评估。拟议工作的成果 将是对丛枝锦鸡儿进行遗传操作的改进工具，这将有利于研究人员使用 这个模型，以及对不同动质体种与其各自的 昆虫寄主。

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.