Extended Data Fig. 8: Mode of uptake and membrane interactions of FA prodrug.
From: Porin-independent accumulation in Pseudomonas enables antibiotic discovery
a) (LEFT) In PBS at 37 °C, FA prodrug (10 µg/ml) hydrolyses to FA (FA) over a period of 48 h, as monitored via LC–MS. TIC LC–MS traces showing the disappearance of FA prodrug and the appearance of FA. Traces are all normalized to 2E6 ion counts. (RIGHT) Plasma stability, microsomal stability and 72-hour IC50 values for FA and FA prodrug. Average percentage errors are reported as s.e.m. IC50 errors reported as s.d. n = 3 biologically independent samples. b) Cotreatment with magnesium ions leads to a 32x increase in MIC for gentamicin and FA prodrug, while FA shows no change. Treatment with 80 mM NaCl shows minimal change when compared to the MgCl2 treated samples. MICs performed in P. aeruginosa PAO1 according to CLSI guidelines and values are reported in µg/ml. n = 3 biologically independent samples c) FA prodrug, gentamicin and colistin all permeabilize the outer-membrane of P. aeruginosa PAO1 to the membrane impermeable fluorophore NPN at a 10-minute time point, while treatment with FA shows no effect. Error bars represent the standard deviation from the average of RFUs. n = 3 biologically independent samples d) Treatment with FA prodrug leads to dose-dependent inner-membrane depolarization in P. aeruginosa PAO1, quantified using the potentiometric dye DiSC3 (5), while treatment with FA shows no effect. Concentrations of FA and FA prodrug are listed in µg/ml. 1% Triton X was used as the positive control, while 2% DMSO was used as the negative control. n = 3 biologically independent samples.
