Dataset Information

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 ?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 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 ?rpoN 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 ?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.

SUBMITTER: Hall CW

PROVIDER: S-EPMC6761562 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Hall Clayton W CW Farkas Eszter E Zhang Li L Mah Thien-Fah TF

Antimicrobial agents and chemotherapy 20190923 10

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 ...[more]

PMID: 31383655

Similar Datasets

Project description:Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.

Dataset Information

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

Publications

Potentiation of Aminoglycoside Lethality by C<sub>4</sub>-Dicarboxylates Requires RpoN in Antibiotic-Tolerant Pseudomonas aeruginosa.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets