Measuring Effector-Mediated Modulation of Inflammatory Responses to Infection with Enteropathogenic and Shiga Toxin-Producing E. coli.

Shiga toxin-producing Escherichia coli (STEC) and the related pathogen enteropathogenic Escherichia coli (EPEC) use a type III secretion system to translocate effector proteins into host cells to modulate inflammatory signaling pathways during infection. Here we describe the procedures to investigate effector-driven modulation of host inflammatory signaling pathways in mammalian cells where bacterial effectors are ectopically expressed or in cell lines infected with STEC or EPEC. We focus on the TNF-induced NF-κB response by examining IκBα degradation by immunoblot and p65 nuclear localization in addition to using an NF-κB-dependent luciferase reporter and cytokine secretion assays. These methods can be adapted for examining effector-mediated modulation of other inflammatory stimuli and host signaling pathways.