Nanostring profiling of urothelial cells intracellularly infected with UPEC

The primary goals of this study are to: determine how intracellular infection of urothelial cells with uropathogenic Escherichia coli influences urothelial cell metabolism, and determine the influence of cytochrome bd on the urothelial cell response to infection HTB-9 urothelial cells were propagated and infected in RPMI (Gibco) supplemented with 10% fetal bovine serum (Gibco). Cells were mock infected or infected either with UPEC isolate UTI89 or an isogenic mutant lacking cytochrome bd (∆cydAB). Bacteria were added to urothelial cell monolayers at a multiplicity of infection (MOI) of 5-10, centrifuged at 600 x g for 5 minutes to facilitate and synchronize attachment, and incubated for two hours at 37˚C in 5% CO2. Monolayers were washed thoroughly with PBS, and fresh RPMI containing 100 µg/mL gentamicin (Gibco) was added to each well to kill extracellular bacteria. Monolayers were incubated another two hours and washed with PBS prior to RNA extraction. RNA was extracted was intracellularly infected urothelial cells using TRIzol Reagent (Invitrogen) according to manufacturer’s protocols. 20 µg nuclease free glycogen (Sigma) was added to facilitate RNA precipitation. 100 ng purified RNA (20 ng/µL) was hybridized for 20 hours and analyzed using the nCounter Human Metabolic Pathways Panel (NanoString Technologies) according to manufacturer’s protocols. RNA was analyzed from at least three biological replicates per condition. Raw data were normalized and subjected to background thresholding using default settings. Normalized data were analyzed using the nCounter Advanced Analysis platform (v2.0.134) plugin for nSolver using default settings (Benjamini-Yekutieli method).