Methods in molecular biology (Clifton, N.J.) 2674 (-) 295-311 [2023-06-01; online 2023-06-01]
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.