Potentiation of Aminoglycoside Lethality by C4-Dicarboxylates Requires RpoN in Antibiotic-Tolerant Pseudomonas aeruginosa

Antibiotic tolerance contributes to the inability of standard antimicrobial therapies to clear the chronic Pseudomonas aeruginosa lung infections that often afflict patients with cystic fibrosis (CF). Metabolic potentiation of bactericidal antibiotics with carbon sources has emerged as a promising strategy to resensitize tolerant bacteria to antibiotic killing. Fumarate (FUM), a C-4-dicarboxylate, has been recently shown to resensitize tolerant P. aeruginosa to killing by tobramycin (TOB), an aminoglycoside antibiotic, when used in combination (TOB+FUM). Fumarate and other C-4-dicarboxylates are taken up intracellularly by transporters regulated by the alternative sigma factor RpoN. Once in the cell, FUM is metabolized, leading to enhanced electron transport chain activity, regeneration of the proton motive force, and increased TOB uptake. In this work, we demonstrate that a Delta rpoN mutant displays impaired FUM uptake and, consequently, nonsusceptibility to TOB+FUM treatment. RpoN was also found to be essential for susceptibility to other aminoglycoside and C-4-dicarboxylate combinations. Importantly, RpoN loss-of-function mutations have been documented to evolve in the CF lung, and these loss-of-function alleles can also result in TOB+FUM nonsusceptibility. In a mixed-genotype population of wild-type and Delta rpoN cells, TOB+FUM specifically killed cells with RpoN function and spared the cells that lacked RpoN function. Unlike C-4-dicarboylates, both o-glucose and L-arginine were able to potentiate TOB killing of Delta rpoN stationary-phase cells. Our findings raise the question of whether TOB+FUM will be a suitable treatment option in the future for CF patients infected with P. aeruginosa isolates that lack RpoN function.