Glycerol metabolism modulates antibiotic tolerance in Pseudomonas aeruginosa
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ABSTRACT:
PROVIDER: PRJNA1177469 | ENA |
REPOSITORIES: ENA
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Project description:Chronic infections with Pseudomonas aeruginosa are a leading cause of morbidity and mortality in persons with cystic fibrosis (pwCF). P. aeruginosa persists in the CF lung by utilizing adaptation strategies to cause infection, including altering the expression of metabolic genes to acquire nutrients that are abundant in the CF airway. Glycerol in the airway is imported and metabolized by the glp regulon, which is under the control of the GlpR repressor. It has been shown that the loss of GlpR results in increased biofilm development in P. aeruginosa CF isolate compared to a wound isolate. Based on the increased biofilm phenotype observed and because biofilms are associated with increased antibiotic tolerance, we questioned whether GlpR plays a role in mediating antibiotic resistance of P. aeruginosa. We measured tobramycin tolerance in wild-type and glpR-defective P. aeruginosa isolates from the CF airway (FRD1) and a wound (PAO1). Cultures were grown in lysogeny broth or synthetic cystic fibrosis sputum consisting of the base formula of primarily amino acids (SCFM1) or supplemented with mucins and DNA (SCFM2), with dose-dependent concentrations of tobramycin. We tested the impact of a glpR mutation on P. aeruginosa adherence on bronchial epithelial cells from pwCF (CFBE) in the presence of tobramycin. CFBE cells were inoculated at an MOI of ~1:20 for 1 hour, given fresh apical media for 5 more hours, then apical and basal media was replaced with media containing 20 µg/ml tobramycin. We measured colony forming units (CFUs) and lactate dehydrogenase (LDH) release for cytotoxicity. Loss of glpR increased tolerance to tobramycin in both the PAO1 and FRD1 backgrounds in vitro at a concentration of 0.625 µg/mL in lysogeny broth and SCFM1. On both CFBE’s and 16HBE’s, the antibiotic resistance phenotype was more prominent in FRD1 glpR with a 2-log increase in viable bacteria when grown on cells and treated with 20 ug/ml tobramycin. However, changes in cytotoxicity where not observed between wildtype and GlpR mutants as LDH measurements were not significantly different. Our results indicate that GlpR may regulate antibiotic tolerance, in addition to biofilm development and glycerol metabolism. Additional studies are necessary to determine the mechanism of how GlpR modulates biofilm development and antibiotic tolerance.
2025-09-05 | GSE280261 | GEO
Project description:Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The superior capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states, and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residues R152, K270, or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG sensing mechanism by RccR.
2023-07-20 | GSE237535 | GEO
Project description:Bacterial persisters are frequently described as metabolically dormant, yet the endogenous metabolic programs that sustain survival during prolonged nutrient limitation remain poorly understood. Here, using stationary-phase Escherichia coli as a model of antibiotic tolerance, we combine proteomics, genetics, metabolic phenotyping, and single-cell imaging to define a metabolic framework underlying persistence. Perturbation of tricarboxylic acid cycle function broadly reprogrammed stationary-phase physiology, suppressing lipid and glycerol metabolism, altering energy homeostasis and proteostasis, and reducing antibiotic tolerance. Systems-level analyses identified phospholipid-derived glycerol catabolism as a central metabolic node linking endogenous carbon recycling to persistence. Genetic disruption of glycerol utilization impaired proton motive force homeostasis, reduced formation of large polar protein aggregates, altered division-associated remodeling, and sensitized cells to antibiotic-induced lysis. Functional metabolic assays further revealed that persisters retain a selective capacity to utilize glycerol for rapid proton motive force restoration without growth resumption. Together, our findings support a model in which stationary-phase persisters are not metabolically inert but sustained through endogenous metabolic rewiring that coordinates energy maintenance, proteostasis, and antibiotic tolerance.
2026-06-12 | PXD079272 | Pride
Project description:Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, using genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1, we identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections. This dataset compares the expression of SAH108, a strain with enhanced antibiotic tolerance in the biofilm state, to expression in wild-type strains. We compared the expression of two biological replicates from strain SAH108 to samples from three wild-type, reference strains. All samples were collected from exponentially-growing planktonic cultures.
2011-11-21 | E-GEOD-26142 | biostudies-arrayexpress
Project description:Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, using genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1, we identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections. This dataset compares the expression of SAH108, a strain with enhanced antibiotic tolerance in the biofilm state, to expression in wild-type strains.
2011-11-21 | GSE26142 | GEO
Project description:Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen. Chronic infections are associated with biofilms, conferring resistance to antimicrobial agents and complicating treatment strategies. This study focuses on understanding the role of the uncharacterized gene PA3049, upregulated under biofilm conditions. In the context of P. aeruginosa biofilms, PA3049 emerged as a player in withstanding antimicrobial challenges both in vitro and in clinically validated infection models. Under antibiotic conditions, the deletion of PA3049 resulted in reduced pyocyanin production and altered abundance of enzymes controlling denitrification, pyoverdine, and hydrogen cyanide biosynthesis. Notably, PA3049 directly interacts with two kinases implicated in antibiotic tolerance, inactivating their active sites. Renamed as the Biofilm antibiotic tolerance Regulator (BatR), PA3049 is a key player in P. aeruginosa biofilm maintenance and antimicrobial tolerance. These findings contribute to understanding the complex bacterial lifestyle in biofilms, shedding light on a previously uncharacterized gene with significant implications for combating multidrug-resistant infections.
2026-06-26 | PXD050995 | Pride
Project description:Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen. Chronic infections are associated with biofilms, conferring resistance to antimicrobial agents and complicating treatment strategies. This study focuses on understanding the role of the uncharacterized gene PA3049, upregulated under biofilm conditions. In the context of P. aeruginosa biofilms, PA3049 emerged as a player in withstanding antimicrobial challenges both in vitro and in clinically validated infection models. Under antibiotic conditions, the deletion of PA3049 resulted in reduced pyocyanin production and altered abundance of enzymes controlling denitrification, pyoverdine, and hydrogen cyanide biosynthesis. Notably, PA3049 directly interacts with two kinases implicated in antibiotic tolerance, inactivating their active sites. Renamed as the Biofilm antibiotic tolerance Regulator (BatR), PA3049 is a key player in P. aeruginosa biofilm maintenance and antimicrobial tolerance. These findings contribute to understanding the complex bacterial lifestyle in biofilms, shedding light on a previously uncharacterized gene with significant implications for combating multidrug-resistant infections.
2026-06-26 | PXD050997 | Pride
Project description:Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics were examined using both quantitative proteomics and transposon sequencing. These screens indicated that arginine metabolism is involved in antibiotic tolerance within a biofilm and led to the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction was found to induce antibiotic tolerance via inhibition of protein synthesis. In a mouse skin infection model, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.
2024-07-10 | GSE267626 | GEO
Project description:Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen, with chronic infections often associated with biofilms that enhance antibiotic resistance. This study investigates the uncharacterized gene PA3049, which is upregulated under biofilm conditions, to determine its role in infection, biofilm formation, and antimicrobial tolerance. o Using bioinformatics, infection models, and molecular microbiology, we determined that PA3049 contributed to biofilm establishment both in vitro and in high-validity infection models. We also identified its role in bacterial survival under sub-inhibitory concentrations of antibiotics and its impact on pyocyanin production. Proteomic analysis revealed that PA3049 upregulates the R2-type pyocin cluster, which drives explosive cell lysis and extracellular DNA (eDNA) release during early stages of P. aeruginosa biofilm development. Additionally, PA3049 interacts with PA0486, an uncharacterized Ser/Thr protein kinase implicated in pyocyanin production and bacterial killing, suggesting a putative in vivo mechanism of action. o Renamed as the Biofilm antibiotic tolerance Regulator (BatR), PA3049 emerges as a key player in P. aeruginosa biofilm maintenance and resistance. These findings provide new insights into bacterial biofilm dynamics and highlight two previously uncharacterized genes with potential implications for combating multidrug-resistant infections
2026-06-26 | PXD062857 | Pride
Project description:Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics were examined using both quantitative proteomics and transposon sequencing. These screens indicated that arginine metabolism is involved in antibiotic tolerance within a biofilm and led to the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction was found to induce antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.
2024-07-11 | PXD052440 | Pride