Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-Lactamase Fusions.
Detection of Cells Translocated with Yersinia Yops in Infected Tissues Using β-Lactamase Fusions.
复制标题
使用β-内酰胺酶融合检测感染组织中耶尔森氏菌易位的细胞。
DOI：
10.1007/978-1-4939-9541-7_9
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Mecsas,Joan
中科院分区：
文献类型：
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作者：
Nguyen,GiangT;McCabe,AnneL;Fasciano,AlyssaC;Mecsas,Joan
基于TEM-CCF2/4-AM FRET系统的开发使研究人员能够追踪感染期间效应蛋白转移到哺乳动物细胞中的情况。这使得易位和非易位细胞群体得以分离，用于进一步研究。Yersiniastrains表达翻译YOP-TEM型融合，包含YOP与β-内酰胺酶TEM1部分的分泌和易位信号，用于感染小鼠、从小鼠分离的组织或培养中的哺乳动物细胞。用CCF2-AM或CCF4-AM处理感染和收获的哺乳动物细胞，并用荧光激活细胞分选(FACS)分析检测这种荧光化合物的切割。单个细胞的绿色发射光谱到蓝色发射光谱的转变表明，在耶尔森尼感染期间，由于化合物的两个荧光之间的FRET中断，给定的YOP-TEM融合蛋白移位到宿主细胞中。在耶尔森氏菌中，这种方法已经被用来了解感染过程中效应器移位的细胞的III型分泌动力学和YOP功能。在这里，我们描述了如何产生YOP-TEM体，以及如何通过细胞分类和流式细胞术分析来检测、定量、分离和研究感染过程中小鼠组织和体外组织中含有YOP-TEMs的细胞。此外，我们还提供了通过平板阅读器和荧光显微镜分析透射电子显微镜阳性细胞的指导。
Development of the TEM-CCF2/4-AM FRET-based system has enabled investigators to track translocation of effector proteins into mammalian cells during infection. This allows for separation of translocated and non-translocated cell populations for further study.Yersiniastrains expressing translational Yop-TEM fusions, containing the secretion and translocation signals of a Yop with the TEM-1 portion of β-lactamase, are used to infect mice, tissues isolated from mice, or mammalian cells in culture. Infected and harvested mammalian cells are treated with either CCF2-AM or CCF4-AM, and cleavage of this fluorescent compound by TEM is detected by fluorescence-activated cell sorting (FACS) analysis. A shift from green to blue emission spectra of individual cells is indicative of translocation of a given Yop-TEM fusion protein into the host cell duringYersiniainfection due to a disruption in FRET between the two fluors of the compound. InYersinia, this method has been used to understand Type III secretion dynamics and Yop functions in cells translocated by effectors during infection. Here, we describe how to generate Yop-TEM constructs, and how to detect, quantify, isolate, and study Yop-TEM containing cells in murine tissues during infection and in ex vivo tissues by cell sorting and flow cytometry analysis. In addition, we provide guidance for analyzing TEM-positive cells via a plate reader and fluorescent microscopy.