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

一些微生物病原体是专性的细胞内病原体。它们很难研究，因为它们复杂的生命周期发生在宿主的细胞内。在生物学的其他领域，一种称为荧光激活细胞分选(FACS)的技术被用于识别和分离不同类型的细胞，从复杂的混合物到近乎均匀的混合，基于差异荧光染色。这项提议将使用这项技术来测量和分离一种感染蜜蜂并对蜂群健康造成负面影响的特定寄生虫(中华微孢子虫)的不同生活阶段。在这项建议资助的工作中，这项技术的使用有望增加对蜜蜂感染过程的了解。这一新系统还可以用来增加对蜜蜂或其他动物细胞内其他病原体生物学的了解。因此，该项目服务于美国国家科学基金会促进科学进步的使命。反过来，使用这项技术的发现可能是确定治愈或预防感染的方法的第一步。因此，这项技术可以用于生物经济学中，研究人员希望稳定蜜蜂种群和授粉。专性细胞内病原体的研究很困难，因为它们不容易在无菌条件下在宿主外繁殖。许多研究方法都要求将病原体的细胞与寄主的细胞分离。此外，要研究具有复杂生活周期的胞内病原菌物种，关键是还可以分离出个体的生活阶段。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是复杂的，对于这些非模式生物，没有或有限的物种特有的分子工具。目前存在的障碍可以使用基于流式细胞仪的技术来相对量化和定位蜜蜂的不同细胞类型，这些细胞类型包含蜜蜂的关键病原体-中华微孢子虫的不同生活期。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体，有望开发出一种新的系统，以促进对宿主-病原体相互作用和感染动力学的了解。这项技术的建立有望促进微孢子虫研究的一些新方向。由于这项技术评估的一些细胞特征在真核生物中广泛存在，因此很有可能该技术可以单独用于来自不同系统发育组的其他病原体物种，或者与其他可用的染料结合使用。这项提议还将培养一名微生物学研究方面的本科生。因此，这项建议是在培训下一代科学工作者。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.