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

有些微生物病原体是专性细胞内病原体。它们很难研究，因为它们在宿主细胞内发生复杂的生命周期。在生物学的其他领域，一种称为荧光活化细胞分选（FACS）的技术被用于识别和分离不同的细胞类型，从复杂的混合物到接近均匀的基于差异荧光染色。该提案将使用该技术来测量和分离感染蜜蜂并对蜂群健康产生负面影响的特定寄生虫（微孔虫）的不同生命阶段。在这项提案资助的工作中，使用这种技术有望增加对蜜蜂感染过程的理解。这个新系统也可以用来增加对蜜蜂或其他动物的其他细胞内病原体的生物学知识。因此，这个项目服务于NSF促进科学进步的使命。反过来，使用这种技术的发现可以成为确定治疗或预防感染方法的第一步。因此，这项技术可以被那些想要稳定蜜蜂种群和授粉的研究人员用于生物经济。专性细胞内病原体的研究是困难的，因为它们在无菌条件下不容易在宿主外繁殖。许多研究途径都需要将病原体的细胞与宿主的细胞分开。此外，研究具有复杂生命周期的胞内病原菌种，也可以对关键的个体生命阶段进行分离。在宿主和病原体都是非模式生物的情况下，这种操作和分离的实验挑战更加复杂，因为只有有限的或没有特定物种的分子工具。这些障碍可以通过基于流式细胞术的技术来解决，该技术可以相对定量和定位蜜蜂不同类型的细胞，这些细胞含有蜜蜂的关键病原体微孢子虫（Nosema ceranae microsporidian）的不同生命阶段。专门的细胞染料测量一般细胞特征，结合流式细胞术分离不同的细胞群，有望开发一个新的系统，以促进对宿主-病原体相互作用和感染动力学的理解。这项技术的建立有望促进微孢子虫研究的一些新方向。由于该技术评估的一些细胞特征在真核生物中广泛存在，因此该技术很有可能适用于来自不同系统发育群的其他病原体物种单独使用或与其他可用染料结合使用。这一建议还将培养一名微生物学研究方面的本科生。因此，这一提议正在培养下一代科学工作者。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.