Reviewer #2 (Public Review): Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

Опубликована: Фев. 14, 2024

Aminoglycosides, a class of antibiotics, have been in use for decades, displaying broad-spectrum activity against Gram-negative and Gram-positive bacteria. They target ribosomes disrupt protein synthesis. Although their declined due to newer antibiotics with lower toxicity, increasing drug resistance has renewed interest aminoglycosides. Herein, we demonstrated that energy metabolism plays crucial role aminoglycoside tolerance, as knockout strains deleted genes associated the tricarboxylic acid cycle (TCA) electron transport chain (ETC) exhibited increased tolerance aminoglycosides mid-exponential growth phase Escherichia coli cells. Our initial hypothesis posited genetic perturbations would lead reduction proton motive force, subsequently affecting uptake This is based on prevailing notion dependent distinctive energy-driven electrochemical potential across cytoplasmic membrane. However, our results did not support this hypothesis. Despite mutant strains, found no consistent metabolic changes, ATP levels, pH variations, or membrane differences compared wild-type strains. Additionally, intracellular concentrations fluorophore-labeled gentamicin remained similar all To uncover mechanism responsible observed employed untargeted mass spectrometry quantify proteins within these mutants them counterparts. comprehensive analysis, which encompassed protein-protein association networks functional enrichment, unveiled noteworthy upregulation linked TCA suggesting compensate perturbation by maintain levels. Furthermore, pathway enrichment analysis shed light local network clusters downregulation were both large small ribosomal binding proteins, ribosome biogenesis, translation factor activity, biosynthesis ribonucleoside monophosphates. These findings offer plausible explanation Altogether, research mechanisms behind paving way novel strategies combat such

Язык: Английский

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eLife assessment: Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance

Опубликована: Фев. 14, 2024

Aminoglycosides, a class of antibiotics, have been in use for decades, displaying broad-spectrum activity against Gram-negative and Gram-positive bacteria. They target ribosomes disrupt protein synthesis. Although their declined due to newer antibiotics with lower toxicity, increasing drug resistance has renewed interest aminoglycosides. Herein, we demonstrated that energy metabolism plays crucial role aminoglycoside tolerance, as knockout strains deleted genes associated the tricarboxylic acid cycle (TCA) electron transport chain (ETC) exhibited increased tolerance aminoglycosides mid-exponential growth phase Escherichia coli cells. Our initial hypothesis posited genetic perturbations would lead reduction proton motive force, subsequently affecting uptake This is based on prevailing notion dependent distinctive energy-driven electrochemical potential across cytoplasmic membrane. However, our results did not support this hypothesis. Despite mutant strains, found no consistent metabolic changes, ATP levels, pH variations, or membrane differences compared wild-type strains. Additionally, intracellular concentrations fluorophore-labeled gentamicin remained similar all To uncover mechanism responsible observed employed untargeted mass spectrometry quantify proteins within these mutants them counterparts. comprehensive analysis, which encompassed protein-protein association networks functional enrichment, unveiled noteworthy upregulation linked TCA suggesting compensate perturbation by maintain levels. Furthermore, pathway enrichment analysis shed light local network clusters downregulation were both large small ribosomal binding proteins, ribosome biogenesis, translation factor activity, biosynthesis ribonucleoside monophosphates. These findings offer plausible explanation Altogether, research mechanisms behind paving way novel strategies combat such

Язык: Английский

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HigA2 (Rv2021c) Is a Transcriptional Regulator with Multiple Regulatory Targets in Mycobacterium tuberculosis

Microorganisms, Год журнала: 2024, Номер 12(6), С. 1244 - 1244

Опубликована: Июнь 20, 2024

Toxin-antitoxin (TA) systems are the major mechanism for persister formation in Mycobacterium tuberculosis (Mtb). Previous studies found that HigBA2 (Rv2022c-Rv2021c), a predicted type II TA system of Mtb, could be activated transcription response to multiple stresses such as anti-tuberculosis drugs, nutrient starvation, endure hypoxia, acidic pH, etc. In this study, we determined binding site HigA2 (Rv2021c), which is located coding region upstream gene higB2 (Rv2022c), and conserved recognition motif was characterized via oligonucleotide mutation. Eight sites were further Mtb genome according motif. RT-PCR showed can regulate level all eight these genes three adjacent downstream genes. DNA pull-down experiments twelve functional regulators sense external regulatory signals may system. Of these, Rv0903c, Rv0744c, Rv0474, Rv3124, Rv2603c, Rv3583c involved regulation stress signals. general, identified target possible HigA2, paved way illustration persistence establishment Mtb.

Язык: Английский

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Enhanced extracellular matrix production provides protection to cell wall-deficient Escherichia coli

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Ноя. 6, 2024

Abstract Escherichia coli -induced recurrent urinary tract infections (rUTIs) present a complicated challenge within the medical field. Most first-line antibiotic treatments primarily target cell-wall synthesis, which can lead to formation of cell wall-deficient cells. To investigate how such cells sustain, we obtained an E. strain capable efficiently proliferating without its wall. One mutations enhanced expression rcsA , encoding important regulator involved in responding envelope stress. RNA sequencing demonstrated upregulation genes associated with production extracellular matrix components, and this increased was confirmed using various imaging techniques. Remarkably, subsequent long-term evolution experiment on revealed further augmentation production, coinciding ability withstand harsh conditions. These findings demonstrate adapts loss wall that synthesis constituents compensate for protective properties Significance The is crucial protection . During bacterial as infections, antibiotics disrupt are commonly prescribed. However, stimulate bacteria still able proliferate despite presence these drugs. Our reveal increases matrix, mechanism found other unicellular organism too. This adaptation allows maintain their structural integrity survive, highlighting potential successful treatment infections.

Язык: Английский

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Polynucleotide Phosphorylase Mediates a New Mechanism of Persister Formation in Escherichia coli

Microbiology Spectrum, Год журнала: 2022, Номер 11(1)

Опубликована: Дек. 8, 2022

Despite the identification of many genes and pathways involved in persistence phenomenon bacteria, mechanisms are not well understood. Here, using Escherichia coli, we identified polynucleotide phosphorylase (PNPase) as a key regulator persister formation. We constructed pnp knockout strain (Δpnp) its complemented exposed them to antibiotics stress conditions. The results showed that, compared with wild-type W3110, Δpnp had significant defects stresses, phenotype was restored upon complementation gene. Transcriptome sequencing (RNA-seq) analysis revealed that 242 (166 upregulated 76 downregulated) were differentially expressed W3110 strain. KEGG these mostly mapped metabolism virulence pathways, which most positively regulated by global cyclic AMP receptor protein (CRP). Correspondingly, transcription level crp gene increased 3.22-fold early stationary phase. further explored indicators cellular strain, double-deletion mutant, transcriptional activity Our indicate PNPase controls negatively regulating operon via targeting 5'-untranslated region transcript. This study reveals mechanism provides novel targets for development drugs against persisters more effective treatment. IMPORTANCE Persisters pose challenges treatment persistent infections. An improved understanding will provide therapeutic important better treatments. Since recent studies tuberculosis drug pyrazinamide have implicated target, this study, addressed possibility might be coli. demonstrates indeed being persistence, formation, suggests new target treating bacterial

Язык: Английский

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Rcs phosphorelay affects the sensitivity of Escherichia coli to plantaricin BM-1 by regulating biofilm formation

Frontiers in Microbiology, Год журнала: 2022, Номер 13

Опубликована: Ноя. 24, 2022

Introduction: Plantaricin BM-1 is a class IIa bacteriocin produced by Lactobacillus plantarum that exerts significant antibacterial activity against many foodborne bacteria. Studies have shown bacteriocins inhibit Gram-positive bacteria via the mannose phosphotransferase system; however, their mechanism of action Gram-negative remains unknown. In this study, we explored through which Rcs phosphorelay affects sensitivity Escherichia coli K12 cells to plantaricin BM-1. Methods and Results: The minimum inhibitory concentrations E. K12, JW5917 (rcsC mutant), JW2204 (rcsD JW2205 (rcsB mutant) were 1.25, 0.59, 1.31, 1.22 mg/ml, respectively. Growth curves showed increased same level as after complementation. Meanwhile, scanning electron microscopy transmission revealed that, under BM-1, appearance did not significantly differ from cells; cell contents reduced plasmolysis shrinkage observed at both ends. Crystal violet staining laser confocal biofilm formation was rcsC mutation, while proteomic analysis identified 382 upregulated 260 downregulated proteins in JW5917. particular, mutation found affect expression related formation, with growth curve assays showing deletion these Discussion: Consequently, speculated may regulate affecting formation. This finding provides new insights.

Язык: Английский

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