Abstract：
Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.
摘要：
细胞内细菌病原体实施多种策略以靶向宿主细胞并建立感染。对于液泡病原体，宿主细胞内含病原体的液泡运动过程，称为细胞内贩运，是病原体存活和感染进展的核心。通常是由分泌的毒力因子介导的过程，这些因子操纵宿主细胞骨架机制，内化的含病原体的液泡运输到复制部位以建立独特的复制生态位，如果适用，向宿主细胞外围的交通，以进行细胞到细胞的传播。因此，包含病原体的液泡的细胞内定位代表了感染进展的基本测量。这里,我们描述了一种基于荧光显微镜的方法来定量评估细菌细胞内定位，以鼠伤寒沙门氏菌感染肠上皮细胞为模型。这种实验方法可以修改以研究不同宿主细胞类型的感染，和各种各样的病原体。该系统还可以用于检查感染的动力学，确定介导宿主贩运的分泌毒力因子，调查病原体作为贩运目标的宿主因素，并评估负责介导表型的毒力因子内的功能域。总的来说，这些工具可以提供对各种细胞内细菌病原体的发病机理的基本见解，和新的宿主因子被劫持以介导感染。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1:宿主细胞的培养和制备备选方案:培养和制备宿主细胞以评估宿主因子对细菌定位的贡献基本方案2:用鼠伤寒沙门氏菌感染上皮细胞基本方案3:用于分析细菌定位的荧光染色基本方案4:细菌定位的荧光显微镜分析。
