EAGER: An innovative approach for quantification and prospective isolation of Nosema ceranae life stages from host cells with potential for application to diverse pathogen species

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Some microbial pathogens are obligate intracellular pathogens. They are hard to study because of their complex life cycles which occur inside the cells of their hosts. In other areas of biology, a technique called fluorescence activated cell sorting (FACS) is used to identify and separate different cell types from a complex mixture to near homogeneity based on differential fluorescent-based staining. This proposal will use this technique to measure and isolate different life stages of a specific parasite (Nosema ceranae) that infects honey bees and negatively impacts colony health. In the work funded by this proposal, use of this technique is expected to increase the understanding of the course of infection in honey bees. This new system can also be used to increase knowledge of the biology of other intracellular pathogens in bees or other animals. AS such, this project serves the NSF mission to promote the progress of science. In turn, findings from using this technique can be the first step in identifying means to cure or prevent infections. Thus, this technology can be used in the bioeconomy by researchers who want to stabilize honey bee populations and pollination.The study of obligate intracellular pathogens is difficult because they cannot easily be reproduced outside the host in axenic conditions. Many avenues of inquiry necessitate that cells of the pathogen be separated from those of the host. In addition, to study intracellular pathogen species with complex life cycles, it is critical individual life stages can also be separated. The experimental challenges of such manipulations and separations are compounded in situations where both host and pathogen are non-model organisms for which there are limited or no species-specific molecular tools. The obstacles presented can be addressed using flow cytometry-based techniques for relative quantification and localization of different cell types from honey bees that contain different life stages of the Nosema ceranae microsporidian, a key pathogen of honey bees. Specialized cell dyes measuring general cell features, in conjunction with flow cytometry to isolate distinct cell populations, are expected to develop a new system to advance the understanding of host-pathogen interactions and infection dynamics. Establishment of this technique promises to facilitate a number of new directions in microsporidia research. As some of the cellular features assessed by this technique are widespread in eukaryotes, there is a high probability that the technique can be adapted for use with other pathogen species from diverse phylogenetic groups alone or in combination with other available dyes. This proposal will also train an undergraduate student in microbiology research. As such, this proposal is training the next generation of the science workforce.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.