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

Trustees Of Indiana University, Bloomington IN

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Abstract

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.