Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
Methods Mol Biol. 2023;2674:295-311. doi: 10.1007/978-1-0716-3243-7_20.
Bacterial host cell invasion has routinely been investigated by gentamicin protection assays, which are laborsome and suffer from pronounced experimental noise. This chapter describes an internally controlled, medium- to high-throughput method that resolves the capacity of multiple Salmonella virulence factor mutant strains to bind and invade host cells. The method, widely applicable to also other pathogens, is based on the combination of consortia of genetically tagged isogenic bacterial strains and a modified gentamicin protection assay. These protocols provide a flexible tool box to stringently quantify host cell binding and invasive properties of different mutants. Moreover, the method can be applied to both infections of cultured host cells and in vivo animal models, providing a comparable genetic readout, which greatly facilitates comparisons across experimental models.
细菌宿主细胞入侵通常通过庆大霉素保护试验来研究，该试验繁琐且存在明显的实验噪声。本章描述了一种内部对照、中高通量的方法，用于测定多种沙门氏菌毒力因子突变菌株结合和入侵宿主细胞的能力。该方法广泛适用于其他病原体，基于遗传标记的同基因细菌菌株的联合体和改良的庆大霉素保护试验。这些方案为严格量化不同突变体的宿主细胞结合和侵袭特性提供了灵活的工具盒。此外，该方法可应用于培养宿主细胞的感染和体内动物模型，提供可比的遗传读数，这极大地促进了实验模型之间的比较。
