Exogenous glutamate potentiates gentamicin to kill multidrug- and carbapenem-resistant Pseudomonas aeruginosa through activating biosynthesis of unsaturated fatty acids
Ontology highlight
ABSTRACT: Antibiotic-resistant Pseudomonas aeruginosa is a major clinical challenge due to limited drug uptake. This study shows that exogenous glutamate restores gentamicin efficacy by reprogramming bacterial metabolism to enhance membrane permeability. The effect is mediated through increased biosynthesis of unsaturated fatty acids, which is further confirmed by oleic acid supplementation. These findings reveal a novel metabolic approach to overcome multidrug and carbapenem resistance, offering a promising adjunct strategy to improve antibiotic treatment outcomes.
INSTRUMENT(S): Gas Chromatography MS - positive
PROVIDER: MTBLS12656 | MetaboLights | 2025-09-22
REPOSITORIES: MetaboLights
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| a_MTBLS12656_GC-MS_positive__metabolite_profiling.txt | Txt | |||
| i_Investigation.txt | Txt | |||
| m_MTBLS12656_GC-MS_positive__metabolite_profiling_v2_maf.tsv | Tabular | |||
| s_MTBLS12656.txt | Txt |
Items per page: 5 1 - 4 of 4 |
Similar Datasets
Project description:<p>The antibiotic susceptibility/resistance of bacteria is influenced by their metabolic states. In tobramycin-resistant Edwardsiella tarda (LTB4-RToB), a dynamic metabolic state is observed, characterized by a general reduction in most metabolites as the minimum inhibitory concentration (MIC) increases. Among these, aspartate downregulation emerges as the most critical biomarker. Exogenous aspartate enhances the efficacy of tobramycin, enabling it to effectively kill both lab-evolved and clinically isolated multidrug-resistant (MDR) E. tarda, as demonstrated in an animal model. Aspartate increases intracellular tobramycin levels in a concentration- and time-dependent manner by promoting drug uptake. Metabolic reprogramming analysis of LTB4-RToB and clinically isolated MDR strain WY-28 reveals that exogenous aspartate restores downregulated metabolites, primarily enhancing alanine, aspartate, and glutamate metabolism; the pyruvate cycle; and glycine, serine, and threonine metabolism. These changes elevate the proton motive force and membrane permeability, thereby increasing tobramycin uptake. The resulting intracellular drug concentration surpasses the bactericidal threshold, effectively overcoming resistance in both LTB4-RroB and clinical MDR E. tarda. These findings identify aspartate as a potent metabolic reprogramming agent that potentiates aminoglycosides by boosting intracellular drug accumulation, offering a strategy to combat antibiotic-resistant E. tarda.</p>
2025-07-31 | MTBLS12725 | MetaboLights
Project description:Natural products represent a rich source for antibiotics addressing versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we select elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant revealed unique insights into the mode of action. Elegaphenone enhanced the killing of intracellular P. aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.
2021-09-08 | PXD010476 | Pride
Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a major global health threat, particularly in healthcare-associated infections. While carbapenemase- and porin-centered mechanisms are well characterized, how subinhibitory carbapenem exposure selects noncanonical adaptive routes remains unclear. Here, we show that subinhibitory meropenem promotes O_x001E_antigen loss in K. pneumoniae, predominantly mediated by insertion sequences (IS), thereby enhancing carbapenem resistance. O_x001E_antigen deficiency rewires metabolism under meropenem pressure, especially glycine, serine, and threonine pathways, dampening reactive oxygen species (ROS) accumulation and limiting oxidative killing; exogenous glycine restores ROS production and meropenem susceptibility. Genomic surveys reveal widespread O_x001E_antigen loss in K. pneumoniae, largely driven by IS, and also in Escherichia coli, and O_x001E_antigen–deficient mutants confirm its role in promoting carbapenem resistance. Importantly, this adaptation entails a trade-off: it improves survival under carbapenem pressure but increases serum susceptibility, destabilizes the capsule, attenuates virulence in murine infection models, and confers collateral sensitivity to aminoglycosides. These findings uncover a previously unrecognized route to carbapenem resistance that links O_x001E_antigen remodeling to metabolic rewiring, offering conceptual and therapeutic leverage points.
2025-09-16 | GSE307523 | GEO
Project description:Conjugative plasmids are the main vehicle for the horizontal spread of antimicrobial resistance (AMR). Although AMR plasmids provide advantages to their hosts under antibiotic pressure, they can also disrupt the cell’s regulatory network, impacting the fitness of their hosts. Despite the importance of plasmid-bacteria interactions on the evolution of AMR, the effects of plasmid carriage on host physiology has remained underexplored, and most studies have focused on model bacteria and plasmids that lack clinical relevance. Here, we analyzed the transcriptional response of 11 clinical enterobacterial strains (2 Escherichia coli, 1 Citrobacter freundii and 8 Klebsiella spp.) and the laboratory-adapted E. coli MG1655 to carriage of pOXA-48, one of the most widely spread carbapenem-resistance plasmids. Our analyses revealed that pOXA-48 produces variable responses on their hosts, but commonly affects processes related to metabolism, transport, response to stimulus, cellular organization and motility. More notably, the presence of pOXA-48 caused an increase in the expression of a small chromosomal operon of unknown function in Klebsiella spp. and C. freundii, which is not present in E. coli. Phylogenetic analysis suggested that this operon has been horizontally mobilized across different Proteobacteria species. We demonstrate that a pOXA-48-encoded LysR transcriptional regulator controls the expression of the operon in Klebsiella spp. and C. freundii. In summary, our results highlight a crosstalk between pOXA-48 and the chromosome of its natural hosts.
2024-07-16 | GSE269852 | GEO
Project description:A series of 16 short proline-rich lipopeptides (SPRLPs) were constructed to mimic longer naturally existing proline-rich antimicrobial peptides. Antibacterial assessment revealed that lipopeptides containing hexadecanoic acid (C16) possess optimal antibacterial activity relative to others with shorter lipid components. SPRLPs were further evaluated for their potential to serve as adjuvants in combination with existing antibiotics to enhance antibacterial activity against drug-resistant Pseudomonas aeruginosa Out of 16 prepared SPRLPs, C12-PRP was found to significantly potentiate the antibiotics minocycline and rifampin against multidrug- and extensively drug-resistant (MDR/XDR) P. aeruginosa clinical isolates. This nonhemolytic C12-PRP is comprised of the heptapeptide sequence PRPRPRP-NH2 acylated to dodecanoic acid (C12) at the N terminus. The adjuvant potency of C12-PRP was apparent by its ability to reduce the MIC of minocycline and rifampin below their interpretative susceptibility breakpoints against MDR/XDR P. aeruginosa An attempt to optimize C12-PRP through peptidomimetic modification was performed by replacing all l- to d-amino acids. C12-PRP demonstrated that it was amenable to optimization, since synergism with minocycline and rifampin were retained. Moreover, C12-PRP displayed no cytotoxicity against human liver carcinoma HepG2 and human embryonic kidney HEK-293 cell lines. Thus, the SPRLP C12-PRP is a lead adjuvant candidate that warrants further optimization. The discovery of agents that are able to resuscitate the activity of existing antibiotics against drug-resistant Gram-negative pathogens, especially P. aeruginosa, is of great clinical interest.
| S-EPMC5914005 | biostudies-literature
Project description:Purpose: Recently we have characterize 43 clinical isolates of Pseudomonas aeruginosa overproducing the efflux pump MexEF-OprN. About 45% of these strains did not display mutations in any of the regulators known to control mexEF-oprN expression. Here we identified a novel regulator of the efflux operon mexEF-oprN that we named CmrA by characterizing in vitro selected mutants from the strain PA14. Methods: MexEF-OprN overproducing spontaneous mutants were selected in vitro using the reference strain PA14 in presence of chloramphenicol. Selected mutants (PJ mutants) were characterized using different criteria such as antibiotic susceptibility and production of virulence traits. Gene expression was evaluated by RT-qPCR. Whole genome sequencing of selected mutants (PJ mutants) was performed using IonTorrent technology. Knock-out mutants were constructed by overlapping PCR and homologous recombination. Finally, a transcriptomic analysis by RNAseq was performed to identify the genes depending on CmrA. Results: Characterization of the 4 selected PJ mutants showed that these strains have resistance and virulence profiles slightly differentent from those typically observed in MexEF-OprN overproducing strains (mexS- NfxC strains). The cmrA+ strains (here called NfxC2) showed moderate resistance to MexEF-OprN antibiotic substrates (ciprofloxacin, chloramphenicol and trimethoprim from 8- to 32-fold compared to PA14) and showed a less compromised capacity to produce some virulence traits (rhamnolipids, elastase, biofilm and pyocyanin) as well as the swarming motility. Whole genome sequencing of PJ mutants led to the identification of SNPs in the gene PA14_38040 (PA14_RS15465 after 2015 genome annotation uptdate). The gene PA14_RS15465 codes for an unknown transcriptional activator and was named cmrA for chlormaphenicol resistance activator. Aminoacid substitutions in CmrA found in PJ mutants were shown to constantly activate this regulator. Further characterization by gene inactivation showed that cmrA is responsible for the activation of the expression of the mexEF-oprN operon in PJ mutants. The locus cmrA was characterized and the complete sequence, including regulatory sequeces, was submitted to GenBank (accession number KX274690). Further analysis showed that the CmrA-activating pathway was dependent on mexS and mexT, both genes are known to affect mexEF-oprN expression. Finally, the transcriptomic analysis of the PJ01 mutant (compared to PA14 and to PJ01∆mexT) revealed that CmrA significally activate the expression of 11 genes most of them coding for enzymes involved in redox regulation. Conclusions: The discovery of cmrA opens the posibility to give an explanation to those clinical strains overproducing the efflux pump MexEF-OprN for unknown reasons. Our data shows that this regulator can activate the efflux pump in a way that the strain is capable to tolerate the antibiotics substrates of the pump and to mantain its capacity to produce some virulence traits. CmrA seems to have a role in the activation of regulatory pathways involved in redox homeostasis as shown by the transcriptomic analysis. The fact that the MexEF-OprN pump is activated at the same time that a redox system let us think that this pump could be related to intracellular detoxification of molecules altering the intracellular homeostasis as it has been previously proposed. We are currently analyzing the transcriptome of CmrA to describe the whole activation pathway of this regulator.
2017-05-01 | GSE86211 | GEO
Project description:A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.
| S-EPMC3719540 | biostudies-literature
Project description:Pseudomonas aeruginosa is a major opportunistic pathogen causing a wide range of infections and one of the most problematic bacteria regarding antibiotic resistance, with an increasing incidence of multidrug and extensively-drug resistant strains, including resistance to last resource antibiotics such as carbapenems. Resistances are often due to complex interplays of naturally and acquired resistance mechanisms which are enhanced by its remarkably large regulatory network. Thus, the use of non-targeted shotgun methodologies such as mass spectrometry-based proteomics is crucial to understand these interplays and to reveal possible strain and species-specific novel mechanisms of antibiotic resistance. The aim of this study was to determine the proteomic response of two carbapenem-resistant and extensively-drug-resistant P. aeruginosa strains to subminimal inhibitory concentrations (sub-MICs) of meropenem. The strains belonged to high-risk clones ST235 and ST395, one carrying a class 1 integron-encoded VIM-4 metallo-β-lactamase and one carrying no acquired antibiotic resistance genes. Each strain was cultivated with three different sub-MICs of meropenem, and a quantitative shotgun proteomic approach was applied, using tandem mass tag (TMT) isobaric labeling followed by nano-liquid chromatography tandem-mass spectrometry, to determine significantly up- or down-regulated proteins and enriched groups of proteins and pathways. Cultivation of both strains with ½ and ¼ of the MIC, resulted in hundreds of differentially regulated proteins, including several β-lactamases, transport-related proteins (including multiple porins and efflux pumps), proteins associated with peptidoglycan metabolism and cell wall organization and dozens of regulatory proteins. Remarkably, all components of the H1 type VI secretion system were up-regulated in one of the strains. Enrichment analyses revealed that multiple metabolic pathways were affected. Additionally, numerous proteins of unknown function were also differentially-regulated in each strain. In conclusion, high subminimal-inhibitory concentrations of meropenem cause massive changes in the proteomes of carbapenem-resistant P. aeruginosa strains, involving a wide range of common and strain-specific mechanisms and proteins, many still uncharacterized which might potentially play a role in the susceptibility of P. aeruginosa to meropenem.
2023-01-05 | PXD034987 | Pride
Project description:The emergence of polymyxin resistance in carbapenem-resistant and extended-spectrum -lactamase (ESBL)-producing bacteria is a critical threat to human health, and new treatment strategies are urgently required. Here, we investigated the ability of the safe-for-human use ionophore PBT2 to restore antibiotic sensitivity in polymyxin-resistant, ESBL-producing, carbapenem-resistant Gram-negative human pathogens. PBT2 was observed to resensitize Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa to last-resort polymyxin class antibiotics, including the less-toxic next-generation polymyxin derivative, FADDI-287. We were unable to select for mutants resistant to PBT2 + FADDI-287 in polymyxin resistant E. coli containing a plasmid-borne mcr-1 gene or K. pneumoniae carrying a chromosomal mgrB mutation. Using a highly invasive K. pneumoniae strain engineered for polymyxin resistance through mgrB mutation, we successfully demonstrated the efficacy of PBT2 + FADDI-287 in vivo for the treatment of Gram-negative sepsis. These data present a new treatment modality to break antibiotic resistance in high priority polymyxin-resistant Gram-negative pathogens.
2020-07-01 | GSE153553 | GEO