Figure 2. Tobramycin Potentiation Requires Increased Respiration through the Electron Transport Chain.

(A) Schematic of the aerobic respiratory apparatus, including glycolysis, TCA cycle, and electron transport chain (ETC) with dehydrogenases (DH), quinone pool, and cytochrome oxidases (CYT). In addition, ETC inhibitors carbonyl cyanide 3-chlorophenylhydrazone (CCCP, blue) and sodium azide (NaN₃, green) are depicted near their targets. Proton motive force (PMF) generated by the ETC drives aminoglycoside uptake through its putative transporter (AmT).

(B) Respiratory activity as quantified by oxygen consumption rate (OCR) in response to treatment of stationary-phase PAO1 cells with 15 mM fumarate (FMR), 30 mM glyoxylate (GLX), or no supplementation. Carbon metabolites were added at 12 min (arrow). Data shown reflect the mean ± SEM (n ≥ 6).

(C) Representative flow cytometry quantification of PMF-dependent membrane potential by DiBAC₄(3) labeling of stationary-phase cells 25 min after incubation either with no metabolite or with 15 mM fumarate or 30 mM glyoxylate. Membrane depolarization expels DiBAC₄(3). Cell counts are normalized to mode. Statistical analysis of geometric means is depicted in Figure S2A.

(D) Effects of ETC inhibition by 50 μM CCCP or 0.1% NaN₃ on fumarate-mediated tobramycin (TBR) resensitization. Survival of stationary-phase cells following 4 hr treatment of 40 mg/L tobramycin with (left) and without (right) 15 mM fumarate supplementation. Values depict the mean ± SEM with significance reported as FDR-corrected p values in comparison with untreated control (CTL): ***p ≤ 0.001 (n = 3).

(E) Representative flow cytometry quantification of the effects of ETC inhibition by 0.1% NaN₃ on fumarate-mediated tobramycin uptake in stationary-phase cells following 15 min treatment with 40 mg/L tobramycin-Texas red and either 15 mM fumarate (FMR) or no supplementation (CTL) (left). NaN₃ treatment inhibits fumarate-induced increases in tobramycin uptake (right). Cell counts are normalized to mode.