Extended Data Fig. 6: DAIA prodrugs increase oxidative stress and cause defects in bacterial respiration, membrane integrity and cell-wall architecture.
From: RETRACTED ARTICLE: IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance
a, b, Respiratory changes in E. coli treated with TPP or with the indicated concentration of the DAIA prodrug C23.28–TPP were compared by measuring OCR (for aerobic respiration) (a) and ECAR (for glycolysis) (b). ***P < 0.001; two-tailed unpaired Student’s t-test, relative to TPP-treated control. c, d, Superoxide (solid line, 2 h after treatment; dotted line, 4 h after treatment) (c) and hydrogen peroxide (d) levels were simultaneously measured by dihydroethidium (DHE) and Amplex red fluorescence respectively. n = 8 biological replicates; data are mean ± s.e.m. e, f, Changes in E. coli membrane integrity, upon TPP or prodrug treatment, measured by Live/Dead (SYTO 9/propidium iodide) assay using flow cytometry (e) or fluorescence microscopy (f). n = 3 biological replicates. Scale bar, 2 μm. g, h, Loss of E. coli membrane potential upon treatment with TPP or prodrug measured by BacLight (DiOC2) assay using flow cytometry (g) or fluorescence microscopy (h). n = 3 biological replicates. Scale bar, 2 μm. i, Scanning electron micrographs (SEM; left) and transmission electron micrographs (TEM; right) compare the morphology of E. coli after 8 h of TPP or prodrug treatment to that of the conditional ispH knockdown E. coli strain CGSC 8074 (ΔispH) kept for 8 h in 1% glucose medium. Red arrows indicate membrane blebbing. j, SEM (top) and TEM (bottom) compare the morphology of V. cholerae after 8 h of TPP or prodrug (C23.28–TPP) treatment. In i, j, images are representative of 20 fields from 3 technical replicates. Scale bar, 400 nm.
