Use of mixed infections to study cell invasion and intracellular proliferation of Salmonella enterica in eukaryotic cell cultures
Use of mixed infections to study cell invasion and intracellular proliferation of Salmonella enterica in eukaryotic cell cultures
复制标题
DOI：
10.1016/j.mimet.2003.09.004
复制
发表时间：
2004-01-01
影响因子：
2.2
通讯作者：
Ramos-Morales, F
中科院分区：
文献类型：
--
作者：
Segura, I;Casadesús, J;Ramos-Morales, F
沙门氏菌上皮细胞系被广泛用作研究沙门氏菌侵袭细胞的体外模型。反过来，吞噬细胞系被用来研究沙门氏菌在细胞内的存活和增殖。我们描述了一种新的方法，从经典的混合感染程序中衍生出来，用于量化肠沙门氏菌血清型鼠伤寒沙门氏菌的入侵和增殖缺陷。真核细胞培养物感染两种菌株(例如，突变型和野生型)。感染后，保留在细胞外的细菌细胞会被庆大霉素清除。在试验结束时，细胞内的细菌被回收并进行培养。然后对每个菌株的菌落进行计数，以计算竞争指数。菌株识别可以通过抗生素耐药性标记或使用编码颜色标记的质粒(例如荧光蛋白)来实现。由于两个菌株在整个过程中暴露在相同的条件下，该程序减少了独立试验之间的可变性，并允许直接测量突变株在入侵或细胞内增殖方面的损害。(C)2003爱思唯尔B.V.保留所有权利。
Epithelial cell lines are widely used as an in vitro model to study cell invasion by Salmonella. In turn, phagocytic cell lines are used to study Salmonella intracellular survival and proliferation. We describe a novel method, derived from the classical mixed infection procedure, to quantify invasion and proliferation defects in Salmonella enterica serovar Typhimurium. A eukaryotic cell culture is infected with two strains (e.g., a mutant and the wild-type). After infection, bacterial cells that remain extracellular are eliminated with gentamicin. At the end of the trial, intracellular bacteria are recovered and plated. Colonies from each strain are then counted for the calculation of a competitive index. Strain discrimination can be achieved either with antibiotic resistance markers or using plasmids encoding color markers (e.g., fluorescent proteins). Because both strains are exposed to the same conditions throughout the process, the procedure decreases the variability between independent trials and allows a direct measurement of the impairment of the mutant in invasion or intracellular proliferation. (C) 2003 Elsevier B.V. All rights reserved.