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

Abstract

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 C₄-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 C₄-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 ΔrpoN 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 C₄-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 ΔrpoN cells, TOB+FUM specifically killed cells with RpoN function and spared the cells that lacked RpoN function. Unlike C₄-dicarboylates, both d-glucose and l-arginine were able to potentiate TOB killing of ΔrpoN 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.

Keywords: Pseudomonas aeruginosa; antibiotic tolerance; fumarate; metabolic potentiation; rpoN; tobramycin.

FIG 1

Stationary-phase ΔrpoN cells are not susceptible to TOB+FUM treatment. (A) Survival of PA14 wild-type and ΔrpoN stationary-phase cells that were incubated for 4 h with or without various concentrations of TOB and/or 15 mM FUM. (B) Survival of PA14 wild-type and ΔrpoN stationary-phase cells that were incubated for 4 h with or without 64 μg/ml TOB and/or various concentrations of FUM. (C) Survival of PA14 wild-type and ΔrpoN stationary-phase cells that were incubated for various periods of time with or without 64 μg/ml TOB and/or 15 mM FUM. (D) Survival of PA14 wild-type and ΔrpoN stationary-phase cells carrying pMQ70 or pMQ70::rpoN following incubation for 4 h with or without 64 μg/ml TOB and/or 15 mM FUM. Data in panels A to D are presented as mean log₁₀ CFU per milliliter ± standard errors of the means (SEM) for at least three biological replicates.

FIG 2

RpoN is required for stationary-phase susceptibility to aminoglycosides combined with C₄-dicarboxylates. Shown are survival data for PA14 wild-type and ΔrpoN stationary-phase cells that were incubated for 4 h with no treatment, various concentrations of an aminoglycoside, 15 mM a C₄-dicarboxylate, or an aminoglycoside with a C₄-dicarboxylate. The tested aminoglycoside and C₄-dicarboxylate combinations were TOB+SUC (A), GEN+FUM (B), and AMK+FUM (C). Data are presented as mean log₁₀ CFU per milliliter ± SEM for three biological replicates.

FIG 3

FUM fails to promote respiration and TOB uptake in ΔrpoN cells. (A) Loss of rpoN impairs FUM uptake. Extracellular [FUM] in cultures of PA14 or ΔrpoN stationary-phase cells carrying pMQ70 or pMQ70::rpoN was measured after 0 or 4 h. Data are presented as mean extracellular [FUM] and SEM for three biological replicates. (B) FUM promotes respiration in wild-type but not ΔrpoN stationary-phase cells. Reduction of resazurin to resorufin was measured over time in PA14 or ΔrpoN stationary-phase cells carrying pMQ70 or pMQ70::rpoN exposed to 0 or 15 mM FUM. Data are shown as mean fluorescence intensity ± SEM for two independent experiments with 3 technical replicates per experiment. Since some data sets were obscured by others on the graph, roman numerals are used to show the locations of the data sets. RFU, relative fluorescence units. (C) PMF is required for TOB+FUM killing. Shown are survival data for stationary-phase cells following 4 h of treatment with or without CCCP and/or TOB+FUM. Data are presented as mean log₁₀ CFU per milliliter ± SEM for three biological replicates. (D) FUM increases intracellular TOB accumulation in wild-type but not ΔrpoN stationary-phase cells. Zones of E. coli DH5α growth inhibition caused by lysates of PA14 or ΔrpoN cells treated with TOB or TOB+FUM were used to approximate relative amounts of intracellular TOB. Data are shown as mean diameters and SEM of the growth inhibition zones for two independent experiments. The dashed line indicates the limit of detection (the diameter of the wells).

FIG 4

RpoN-independent DctA expression restores TOB+FUM susceptibility in ΔrpoN stationary-phase cells. (A) Extracellular [FUM] in cultures of PA14 or ΔrpoN stationary-phase cells carrying pMQ70 or pMQ70::dctA was measured after 0 or 4 h. Data are presented as mean extracellular [FUM] and SEM for three biological replicates. (B) Reduction of resazurin to resorufin was measured over time in PA14 or ΔrpoN stationary-phase cells carrying pMQ70 or pMQ70::dctA exposed to 0 or 15 mM FUM. Data are shown as mean fluorescence intensities ± SEM for two independent experiments with 3 technical replicates per experiment. Since some data sets were obscured by others on the graph, roman numerals are used to indicate the locations of the data sets. (C) Survival of PA14 wild-type and ΔrpoN stationary-phase cells carrying pMQ70 or pMQ70::dctA following incubation for 4 h with or without 64 μg/ml TOB and/or 15 mM FUM. Data are presented as mean log₁₀ CFU per milliliter ± SEM for three biological replicates.

FIG 5

Loss-of-function RpoN alleles from CF clinical isolates impair TOB+FUM killing. (A) Schematic representation of RpoN showing some mutations in the DNA binding domain that have been previously observed in CF clinical isolates. aa, amino acids. (B) Survival of PA14 wild-type and ΔrpoN stationary-phase cells carrying pMQ70, pMQ70::rpoN, or pMQ70::rpoN with clinically relevant point mutations following incubation for 4 h with or without 64 μg/ml TOB and/or 15 mM FUM. Data are presented as mean log₁₀ CFU per milliliter ± SEM for three biological replicates.

FIG 6

TOB+FUM specifically kills cells with RpoN function in mixed-genotype populations. (A to E) PA14-lacZ and ΔrpoN stationary-phase cells were mixed at 1:100 (A), 1:10 (B), 1:1 (C), 10:1 (D), and 100:1 (E) ratios, and survival of individual genotypes was determined prior to treatment (0 h) or following 4 h of incubation with either no treatment or TOB+FUM (64 μg/ml TOB and 15 mM FUM). Data are presented as mean log₁₀ CFU per milliliter ± SEM for three biological replicates. (F) Data in panels A to E were used to calculate log₁₀ PA14-lacZ/ΔrpoN ratios of the populations. TOB+FUM treatment consistently reduced the log₁₀ PA14-lacZ/ΔrpoN ratios, indicating a decrease in PA14-lacZ cells relative to ΔrpoN cells in the population.

FIG 7

l-Arginine or d-glucose can potentiate TOB killing of ΔrpoN cells. Shown are survival data for PA14 wild-type and ΔrpoN stationary-phase cells following incubation for 4 h with or without 64 μg/ml TOB in the presence or absence of 10 mM l-arginine (ARG) or 10 mM d-glucose (GLC). Data are presented as mean log₁₀ CFU per milliliter ± SEM for three biological replicates.

FIG 8

Proposed model for RpoN-dependent susceptibility to TOB+FUM. (Left) In the wild-type RpoN⁺ strain, FUM enters the cell through C₄-dicarboxylate transporters (e.g., DctA) that are transcriptionally regulated by RpoN. Once in the cell, FUM is metabolized in the TCA cycle to produce reducing equivalents that promote ETC activity. Increased respiration leads to regeneration of the membrane potential, TOB uptake, and subsequent TOB-dependent cell death. (Right) On the other hand, in the ΔrpoN RpoN⁻ strain, the lack of functional RpoN means that C₄-dicarboxylate transporters are not expressed, and FUM cannot enter the cell. As a consequence, TOB uptake is not increased, and the cell remains tolerant to TOB.