PDF(Pubmed)
Abstract：
Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen-mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention. Key features • Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model. • Live microscopy-based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification. • Assay can be adapted to a broad array of pathogens and diverse host cell types. • Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.
摘要：
细胞内细菌病原体已经进化为善于操纵宿主细胞功能以获得病原体的利益。通常通过分泌的毒力因子靶向宿主途径进行调节。溶酶体途径是细胞内病原体的重要细胞应答途径,因此，代表细菌介导的逃避的常见靶标。这里,我们描述了一种定量评估细菌病原体介导的抑制宿主细胞运输到溶酶体的方法，以鼠伤寒沙门氏菌感染肠上皮细胞为模型。此活细胞成像测定涉及使用BSA的BODIPYTR-X缀合物（DQ-RedBSA），该缀合物在功能性溶酶体中传播并发出荧光。该方法可以适用于研究多种宿主细胞类型中多种病原体的感染。它能够应用于鉴定负责目的表型的分泌的毒力因子以及细菌蛋白内对于介导表型重要的结构域。总的来说，这些工具可以为各种病原菌的发病机制提供宝贵的见解，有可能发现可能是治疗干预的合适靶标的毒力因子。关键特征•基于感染的细菌介导抑制宿主向溶酶体运输的分析，以鼠伤寒沙门氏菌感染人上皮细胞为模型。•基于活显微镜的分析允许单独感染的宿主细胞的可视化,并且适于表型定量。•测定可以适应广泛的病原体和不同的宿主细胞类型。•测定可以鉴定介导表型的毒力因子和介导表型的蛋白质结构域。
