A Multi-Fluorophore Staining Scheme for Identification and Quantification of Vomocytosis.
A Multi-Fluorophore Staining Scheme for Identification and Quantification of Vomocytosis.
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DOI：
10.1021/cbmi.3c00050
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发表时间：
2023-11-27
期刊：
影响因子：
--
通讯作者：
Lewis, Jamal S
中科院分区：
文献类型：
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作者：
Pacifici, Noah;Rojalin, Tatu;Carney, Randy P;Lewis, Jamal S
血细胞增多症是真菌病原体，如新生隐球菌(CN)，在吞噬后从吞噬细胞的消化溶菌体中逃逸的过程。有趣的是，这种排泄使病原体和吞噬细胞都没有受到伤害，并被认为是CNS在受感染宿主中传播的重要机制。这一现象是在2006年发现的，到目前为止，研究几乎完全依赖于通过人工计数延时显微镜视频中的呕吐事件进行量化。这种陈旧的方法有明显的缺点，需要大量的人工分析，吞吐能力有限，并且由于主观性而精度较低。在这里，我们提出了一种测量呕吐细胞率的替代方法，使用多荧光载体报告系统，该系统由感染过程中的两个原位染色步骤和流式细胞仪读数组成。这种方法克服了传统时间推移显微镜方法的局限性，具有处理能力高、操作步骤简单和客观读数准确等关键优势。本研究通过荧光显微镜、共聚焦显微镜和流式细胞术对CN感染的MΦ和DC培养中的呕吐细胞增多报告系统进行了严格的表征。在这里，这个荧光工具被用来观察吞噬小体修饰药物处理后的排泄率的差异，并另外被用来通过拉曼光谱区分荧光激活的细胞分类真菌种群之间的生化成分的差异。此外，该报告器方案被证明适用于测量潜在的生物材料颗粒抛出事件。最终，本文提出的荧光报告系统为测量吞噬物质的呕吐细胞率提供了一个通用工具。这种简便的方法为以前不可行的与呕吐细胞增多相关的研究打开了大门，例如高通量处理、机械筛选和排出物质的下游表征。
Vomocytosis is a process by which fungal pathogens, for instance, Cryptococcus neoformans (CN), escape from the digestive phagolysosome of phagocytic cells after ingestion. Interestingly, this expulsion leaves both the pathogen and phagocyte unharmed, and is believed to be an important mechanism by which CNs disseminate throughout infected hosts. This phenomenon was discovered in 2006, and research to date has relied almost entirely on quantification via manual counting of vomocytosis events in time-lapse microscopy videos. This archaic method has the significant disadvantages of requiring excessive labor in manual analysis, limited throughput capabilities, and low accuracy due to subjectivity. Here, we present an alternative method to measure vomocytosis rates using a multi-fluorophore reporter system comprised of two in situ staining steps during infection and a flow cytometry readout. This approach overcomes the limitations of conventional time lapse microscopy methods, with key advantages of high throughput capability, simple procedural steps, and accurate objective readouts. This study rigorously characterizes this vomocytosis reporter system in CN-infected MΦ and DC cultures via fluorescence microscopy, confocal microscopy, and flow cytometry. Here, this fluorescent tool is used to observe differences in expulsion rates after phagosome-modifying drug treatments and additionally utilized to distinguish differences in biochemical compositions among fluorescence-activated cell sorted fungal populations via Raman spectroscopy. Furthermore, this reporter scheme is demonstrated to be adaptable for use in measuring potential biomaterial particle expulsion events. Ultimately, the fluorescent reporter system presented here provides a universal tool for vomocytosis rate measurement of phagocytosed material. This facile approach opens the door to previously unfeasible types of vomocytosis-related studies such as high throughput treatment mechanistic screening and downstream characterization of expelled material.