Fig 1.

MdrM transporter function is required for activation of the IFN-β response during macrophage infection. (A) Western blot analysis of bacterial membrane fraction probed for 6×His-tagged MdrM and MdrM-F58V (using HisProbe-HPR) expressed from the IPTG-inducible vector pLIV2, with and without IPTG. (B) Growth in the presence of R6G (3.5 μM) of WT L. monocytogenes (L.m.), the ΔmdrM mutant, and the ΔmdrM mutant harboring pLIV2 expressing MdrM-6×His or MdrM-F58V-6×His, with and without IPTG (1 mM) in BHI. The experiment was performed in a 96-well format in a Synergy HT Biotek plate reader. Growth curves from one representative experiment are shown. Error bars representing the standard deviation of a triplicate sample are hidden by the symbols. (C) Intracellular growth curves of WT L. monocytogenes, the ΔmdrM mutant, or the ΔmdrM mutant harboring pLIV2 expressing MdrM-6×His or MdrM-F58V-6×His, with and without IPTG, in RAW264 macrophages. Representative growth curves are shown. Error bars represent standard deviations of 3 biological repeats. (D) RT-qPCR analysis of IFN-β transcriptional levels in macrophages infected with WT L. monocytogenes, the ΔmdrM mutant, or the ΔmdrM mutant harboring pLIV2 expressing MdrM-6×His or MdrM-F58V-6×His, with and without IPTG, at 6 h postinfection (p.i.). Transcription levels are represented as relative quantity (RQ) compared to levels in uninfected cells (un). The data are an average of 3 independent experiments. Error bars represent 95% confidence intervals. *, P < 0.01 compared to the rest of the samples. The data in panels A to C are representative of at least 3 independent biological repeats.