Elucidation of the first interspecies chemical signaling mechanisms in Capsaspora owczarzaki--the predator of a human pathogen and a model for the evolution of animal multicellularity

摘要/摘要 原生生物经常与被忽视的传染病有关，他们教会了我们进化的 多细胞生命的起源和宿主-微生物组的相互作用。母项目支持我们的研究 揭示原生孢子中驱动种间化学信号传递的分子和机制 Owczarzaki及其近亲。卡萨斯孢菌自然存在于传播病毒的蜗牛媒介中。 引起血吸虫病的寄生虫。卡萨斯孢子虫捕杀血吸虫，使其成为一种潜在的 防治这种被忽视的热带病的生防剂。然而，没有人知道是哪种分子 Capsaspora感觉到它的蜗牛宿主定居，也不知道它如何感觉到血吸虫的猎物。此外， 卡萨斯孢子虫和其他单细胞全息虫是现存动物最接近的近亲，它们与它们共享 信号和黏附基因。因此，这些原生生物是研究如何在系统发育上相关的模型 动物体内的多细胞表型和微生物共生体在健康和疾病状态下进化并发挥作用。 作为物种间相互作用的生物化学专家，我们正在对分子和 驱动卡萨斯孢菌与蜗牛和血吸虫相互作用的机制，以及 与邻近微生物相关的原生生物。 我们研究的大多数信号诱导表型都需要时间推移成像。 此外，我们依赖于在实验中测试数十到数千种不同的条件 生物测定引导的代谢物混合物的分级，药物抑制剂的剂量-反应分析， 以及突变体库的筛选。考虑到这个项目上几位研究人员的这些需求，我请求 带有BioSpa孵化器/调度器的BioTek Cytation C10成像仪。这个乐器取代了一个退役的高音- 不再可用的内容成像器。所要求的仪器是为介质而明确设计的- 我们所需要的吞吐量活细胞时程成像。它将配备相位和共焦 荧光光学，它将包括用于快速分析细胞聚集、迁移和 荧光。它将具有8个微滴定板的同时延时成像功能，这将防止 导致实验延迟的日程安排冲突。此外，它还将允许单个用户执行大规模 屏幕是我们目前的仪器不可能实现的。此外，通过减少手动花费的时间 收集图像，我们可以花更多的时间来设计实验和发展假设。最后， 该仪器的自动化性质将增加研究人员之间实验的重复性。这 设备将是所有从事这项研究的研究人员必不可少的主要工具。它将会扩大 并加速我们的化学信号发现，这可能会为使用一种新的生防剂和 揭示动物体内受调控的多细胞的核心机制。

Summary/Abstract Protists are frequently involved in neglected infectious diseases, and they teach us about the evolutionary origins of multicellular life and host-microbiome interactions. The parent project supports our studies to uncover the molecules and mechanisms that drive interspecies chemical signaling in the protist Capsaspora owczarzaki and its close relatives. Capsaspora is naturally found in the snail vectors that transmit the parasites that cause schistosomiasis. Capsaspora hunts and kills schistosomes, making it a potential biocontrol agent against this neglected tropical disease. However, no one knows which molecules Capsaspora senses to colonize its snail host, nor how it senses its schistosome prey. Furthermore, Capsaspora and other unicellular holozoans are the closest living relatives of animals, with which they share signaling and adhesion genes. Therefore, these protists are phylogenetically relevant models to study how multicellular phenotypes and microbial symbioses in animals evolved and act in healthy and disease states. As specialists in the biochemistry of interspecies interactions, we are characterizing the molecules and mechanisms that drive Capsaspora’s interactions with snails and schistosomes, as well the interactions of related protists with neighboring microbes. Time-lapse imaging is required to investigate most of the signal-induced phenotypes that we study. Furthermore, we rely on testing dozens to thousands of different conditions in experiments employing bioassay-guided fractionation of metabolite mixtures, dose-response assays of pharmacological inhibitors, and screening of mutant libraries. Given these needs of several researchers on this project, I am requesting a BioTek Cytation C10 imager with BioSpa incubator/scheduler. This instrument replaces a retired high- content imager that is no longer serviceable. The requested instrument is explicitly designed for the medium- throughput live-cell time-course imaging that we require. It will be equipped with phase and confocal fluorescence optics, and it will include software for rapid analysis of cell aggregation, migration, and fluorescence. It will feature simultaneous time-lapse imaging of eight microtiter plates, which will prevent scheduling conflicts that delay experiments. Additionally, it will allow single users to perform large-scale screens that are impossible with our current instruments. Furthermore, by spending less time manually collecting images, we can devote more time to designing experiments and developing hypotheses. Finally, the automated nature of the instrument will increase reproducibility of experiments between researchers. This equipment will be an essential workhorse instrument for all researchers working on this grant. It will expand and accelerate our chemical signaling discoveries that may both inform the use of a new biocontrol agent and reveal core mechanisms of regulated multicellularity in animals.