Phase separation of RNF214 promotes the progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and the expression and function of an uncharacterized protein RNF214 in HCC are still unknown. Phase separation has recently been observed to participate in the progression of HCC. In this study, we investigated the expression, function, and phase separation of RNF214 in HCC. We found that RNF214 was highly expressed in HCC and associated with poor prognosis. RNF214 functioned as an oncogene to promote the proliferation, migration, and metastasis of HCC. Mechanically, RNF214 underwent phase separation, and the coiled-coil (CC) domain of RNF214 mediated its phase separation. Furthermore, the CC domain was necessary for the oncogenic function of RNF214 in HCC. Taken together, our data favored that phase separation of RNF214 promoted the progression of HCC. RNF214 may be a potential biomarker and therapeutic target for HCC.

Adenosine Monophosphate as a Metabolic Adjuvant Enhances Antibiotic Efficacy against Drug-Resistant Bacterial Pathogens.

Global bacterial infections are on the rise, and drug resistance to bacteria is gradually rendering existing antibiotics ineffective. Therefore, the discovery of new strategies is urgently needed. Cellular metabolism is a key factor in the regulation of bacterial drug resistance, which cannot be separated from the utilization of energetic substances, suggesting that energetic substances may be associated with bacterial drug resistance. In this study, we found that adenosine monophosphate (AMP) can enhance the bactericidal effect of gentamicin against gentamicin-resistant Staphylococcus aureus. This synergistic effect can be generalized for use with different antibiotics and Gram-positive or Gram-negative bacteria. We also validated that the mechanism of AMP reversal of antibiotic resistance involves enhancing the proton motive force via the tricarboxylic acid cycle to increase antibiotic uptake. Simultaneously, AMP increases oxidative stress-induced cell death. This study presents a strategy for adopting low-dose antibiotics to control drug-resistant bacteria, which is important for future drug development and bacterial control.

Upregulated Palmitoleate and Oleate Production in Escherichia coli Promotes Gentamicin Resistance.

Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli. Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli. Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli. Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli. Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli, and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli. This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria.

Exogenous NADH promotes the bactericidal effect of aminoglycoside antibiotics against Edwardsiella tarda.

The global surge in multidrug-resistant bacteria owing to antibiotic misuse and overuse poses considerable risks to human and animal health. With existing antibiotics losing their effectiveness and the protracted process of developing new antibiotics, urgent alternatives are imperative to curb disease spread. Notably, improving the bactericidal effect of antibiotics by using non-antibiotic substances has emerged as a viable strategy. Although reduced nicotinamide adenine dinucleotide (NADH) may play a crucial role in regulating bacterial resistance, studies examining how the change of metabolic profile and bacterial resistance following by exogenous administration are scarce. Therefore, this study aimed to elucidate the metabolic changes that occur in Edwardsiella tarda (E. tarda), which exhibits resistance to various antibiotics, following the exogenous addition of NADH using metabolomics. The effects of these alterations on the bactericidal activity of neomycin were investigated. NADH enhanced the effectiveness of aminoglycoside antibiotics against E. tarda ATCC15947, achieving bacterial eradication at low doses. Metabolomic analysis revealed that NADH reprogrammed the ATCC15947 metabolic profile by promoting purine metabolism and energy metabolism, yielding increased adenosine triphosphate (ATP) levels. Increased ATP levels played a crucial role in enhancing the bactericidal effects of neomycin. Moreover, exogenous NADH promoted the bactericidal efficacy of tetracyclines and chloramphenicols. NADH in combination with neomycin was effective against other clinically resistant bacteria, including Aeromonas hydrophila, Vibrio parahaemolyticus, methicillin-resistant Staphylococcus aureus, and Listeria monocytogenes. These results may facilitate the development of effective approaches for preventing and managing E. tarda-induced infections and multidrug resistance in aquaculture and clinical settings.

Dorsomorphin attenuates ABCG2-mediated multidrug resistance in colorectal cancer.

Colorectal cancer is a common malignant tumor with high mortality, for which chemotherapy resistance is one of the main reasons. The high expression of ABCG2 in the cancer cells and expulsion of anticancer drugs directly cause multidrug resistance (MDR). Therefore, the development of new ABCG2 inhibitors that block the active causes of MDR may provide a strategy for the treatment of colorectal cancer. In this study, we find that dorsomorphin (also known as compound C or BML-275) potently inhibits the transporter activity of ABCG2, thereby preserving the chemotherapeutic agents mitoxantrone and doxorubicin to antagonize MDR in ABCG2-overexpressing colorectal cancer cells. Additionally, dorsomorphin does not alter ABCG2 protein expression. The results of molecular docking studies show that dorsomorphin is bound stably to the ABCG2-binding pocket, suggesting that dorsomorphin is a potent ABCG2 inhibitor that attenuates ABCG2-mediated MDR in colorectal cancer.

Metabolomics analysis of the lactobacillus plantarum ATCC 14917 response to antibiotic stress.

BACKGROUND: Lactobacillus plantarum has been found to play a significant role in maintaining the balance of intestinal flora in the human gut. However, it is sensitive to commonly used antibiotics and is often incidentally killed during treatment. We attempted to identify a means to protect L. plantarum ATCC14917 from the metabolic changes caused by two commonly used antibiotics, ampicillin, and doxycycline. We examined the metabolic changes under ampicillin and doxycycline treatment and assessed the protective effects of adding key exogenous metabolites. RESULTS: Using metabolomics, we found that under the stress of ampicillin or doxycycline, L. plantarum ATCC14917 exhibited reduced metabolic activity, with purine metabolism a key metabolic pathway involved in this change. We then screened the key biomarkers in this metabolic pathway, guanine and adenosine diphosphate (ADP). The exogenous addition of each of these two metabolites significantly reduced the lethality of ampicillin and doxycycline on L. plantarum ATCC14917. Because purine metabolism is closely related to the production of reactive oxygen species (ROS), the results showed that the addition of guanine or ADP reduced intracellular ROS levels in L. plantarum ATCC14917. Moreover, the killing effects of ampicillin and doxycycline on L. plantarum ATCC14917 were restored by the addition of a ROS accelerator in the presence of guanine or ADP. CONCLUSIONS: The metabolic changes of L. plantarum ATCC14917 under antibiotic treatments were determined. Moreover, the metabolome information that was elucidated can be used to help L. plantarum cope with adverse stress, which will help probiotics become less vulnerable to antibiotics during clinical treatment.

Rapid detection of hypobromous acid by a tetraphenylethylene-based turn-on fluorescent AIE probe and its applications.

BACKGROUND: Similar to hypochlorous acid (HClO), hypobromous acid (HBrO) is one of the most notable reactive oxygen species (ROS). Overexpression of HBrO is linked to various diseases causing organ and tissue loss. Due to HBrO's role in the oxidation of micropollutants, real-time monitoring of HBrO in water-based systems is essential. Tetraphenylethylene (TPE)-based organic aggregation-induced emission luminophores (AIEgens) are an emerging category of fluorescent probe materials that have attracted considerable attentions. However, AIE probes are rarely applied to detect HBrO. Developing faster, more precise, and more sensitive AIE probes is thus crucial for detecting biological and environmental HBrO. RESULTS: A small molecule fluorescent probe 4-(1,2,2-triphenylvinyl)benzamidoxime (SWJT-21) was synthesized for the sensitive and selective detection of hypobromous acid (HBrO) based on aggregation-induced emission (AIE). The amidoxime unit of SWJT-21 would undergo an oxidation reaction with HBrO, leading to a structure differentiation between the probe and the product, and therefore the turn-on fluorescence by the AIE effect. The probe could recognize hypobromous acid rapidly (less than 3 s) in high aqueous phase (99 % water) with a turn-on fluorescence response. It was determined that the limit of detection for HBrO was 5.47 nM. Moreover, SWJT-21 demonstrates potential as a test strip for the detection of HBrO. SWJT-21 was also successfully used for the monitoring of HBrO in water samples and for the detection of endogenous/exogenous HBrO in living cells and zebrafish. SIGNIFICANCE: A special AIE fluorescence turn-on probe SWJT-21 based on tetraphenylethylene was designed for detecting HBrO in the environmental and biological systems. This probe has an extremely low detection limit of 5.47 nM and is able to detect HBrO in 99 % aqueous phase in less than 3 s.

Continuous selenite biotransformation and biofuel production by marine diatom in the presence of fulvic acid.

In this study, the biochemical response of Phaeodactylum tricornutum to varying concentrations of inorganic selenium (Se) was investigated. It was observed that, when combined with fulvic acid, P. tricornutum exhibited enhanced uptake and biotransformation of inorganic Se, as well as increased microalgal lipid biosynthesis. Notably, when subjected to moderate (5 and 10 mg/L) and high (20 and 40 mg/L) concentrations of selenite under fulvic acid treatment, there was a discernible redirection of carbon flux towards lipogenesis and protein biosynthesis from carbohydrates. In addition, the key parameters of microalgae-based biofuels aligned with the necessary criteria outlined in biofuel regulations. Furthermore, the Se removal capabilities of P. tricornutum, assisted by fulvic acid, were coupled with the accumulation of substantial amounts of organic Se, specifically SeCys. These findings present a viable and successful approach to establish a microalgae-based system for Se uptake and biotransformation.

Machine Learning-Assisted Portable Microplasma Optical Emission Spectrometer for Food Safety Monitoring.

To meet the needs of food safety for simple, rapid, and low-cost analytical methods, a portable device based on a point discharge microplasma optical emission spectrometer (µPD-OES) was combined with machine learning to enable on-site food freshness evaluation and detection of adulteration. The device was integrated with two modular injection units (i.e., headspace solid-phase microextraction and headspace purge) for the examination of various samples. Aromas from meat and coffee were first introduced to the portable device. The aroma molecules were excited to specific atomic and molecular fragments at excited states by room temperature and atmospheric pressure microplasma due to their different atoms and molecular structures. Subsequently, different aromatic molecules obtained their own specific molecular and atomic emission spectra. With the help of machine learning, the portable device was successfully applied to the assessment of meat freshness with accuracies of 96.0, 98.7, and 94.7% for beef, pork, and chicken meat, respectively, through optical emission patterns of the aroma at different storage times. Furthermore, the developed procedures can identify beef samples containing different amounts of duck meat with an accuracy of 99.5% and classify two coffee species without errors, demonstrating the great potential of their application in the discrimination of food adulteration. The combination of machine learning and µPD-OES provides a simple, portable, and cost-effective strategy for food aroma analysis, potentially addressing field monitoring of food safety.

GLUL stabilizes N-Cadherin by antagonizing ß-Catenin to inhibit the progresses of gastric cancer.

Glutamate-ammonia ligase (GLUL, also known as glutamine synthetase) is a crucial enzyme that catalyzes ammonium and glutamate into glutamine in the ATP-dependent condensation. Although GLUL plays a critical role in multiple cancers, the expression and function of GLUL in gastric cancer remain unclear. In the present study, we have found that the expression level of GLUL was significantly lower in gastric cancer tissues compared with adjacent normal tissues, and correlated with N stage and TNM stage, and low GLUL expression predicted poor survival for gastric cancer patients. Knockdown of GLUL promoted the growth, migration, invasion and metastasis of gastric cancer cells in vitro and in vivo, and vice versa, which was independent of its enzyme activity. Mechanistically, GLUL competed with ß-Catenin to bind to N-Cadherin, increased the stability of N-Cadherin and decreased the stability of ß-Catenin by alerting their ubiquitination. Furthermore, there were lower N-Cadherin and higher ß-Catenin expression levels in gastric cancer tissues compared with adjacent normal tissues. GLUL protein expression was correlated with that of N-Cadherin, and could be the independent prognostic factor in gastric cancer. Our findings reveal that GLUL stabilizes N-Cadherin by antagonizing ß-Catenin to inhibit the progress of gastric cancer.

Automatic and Matrix Interference-Free Acid-Base Titration by Coupling CO2 Vapor Generation with Microplasma Carbon Optical Emission Spectrometry.

Acid-base titration of complex samples is conducive to the rapid evaluation of the degree and risk of environmental pollution to some extent. However, the traditional titration methods usually suffer from serious interference. Herein, an automatic acid-base titration method coupling miniature point discharge optical emission spectrometry (µPD-OES) with CO2 vapor generation was described for the precise, sensitive, and matrix interference-free acid-base titration of complex samples, particularly those with high color intensity, salinity, and turbidity such as wastewater and soil samples. In this work, acid-base titration was carried out in a chemical vapor generator where CO2 was generated through the addition of HCl or NaHCO3, thus enabling efficient separation of CO2 from a complex matrix. The generated CO2 was subsequently swept into the miniaturized point discharge for excitation and further detection by µPD-OES, where the carbon atomic emission at 193.0 nm was monitored. According to the consumed volume and concentration of HCl, accurate and automatic measurements of OH-, CO32-, and HCO3- can be accomplished. The proposed method possesses a high sensitivity of µPD-OES for the detection of CO2 with a relative standard deviation of below 3.0%. Moreover, the proposed system not only retains several unique advantages of accuracy, simplicity, and elimination of the use of complicated, expensive, and high power-consumption instruments but also alleviates the color and turbid interference from complex samples such as dyeing wastewater samples, oilfield water samples, and soil samples. It retains a promising potential application for titration analysis of other samples such as sludge, sediment, and landfill leachate.

L-glutamine sensitizes Gram-positive-resistant bacteria to gentamicin killing.

IMPORTANCE: Methicillin-resistant Staphylococcus aureus (MRSA) infection severely threatens human health due to high morbidity and mortality; it is urgent to develop novel strategies to tackle this problem. Metabolites belong to antibiotic adjuvants which improve the effect of antibiotics. Despite reports of L-glutamine being applied in antibiotic adjuvant for Gram-negative bacteria, how L-glutamine affects antibiotics against Gram-positive-resistant bacteria is still unclear. In this study, L-glutamine increases the antibacterial effect of gentamicin on MRSA, and it links to membrane permeability and pH gradient (ΔpH), resulting in uptake of more gentamicin. Of great interest, reduced reactive oxygen species (ROS) by glutathione was found under L-glutamine treatment; USA300 becomes sensitive again to gentamicin. This study not only offers deep understanding on ΔpH and ROS on bacterial resistance but also provides potential treatment solutions for targeting MRSA infection.

Solid Phase Photothermo-Induced Chemical Vapor Generation: A New Desorption Method for Mercury Analysis by High-Throughput 20-Fiber Direct Immersion Solid Phase Microextraction.

A simple solid phase photothermo-induced chemical vapor generation (SP-PT-CVG) is described and used as an environmentally friendly desorption method for the sensitive determination of mercury in water by direct immersion solid phase microextraction (DI-SPME) atomic fluorescence spectrometry (AFS). A DI-SPME array equipped with 20 nano-TiO2-coated tungsten fibers was employed to simultaneously preconcentrate mercury from 20 samples, enabling an extraction throughput of 40 samples per hour. Subsequently, the fibers were drawn from the sample solutions and inserted into an inner tube sealed in a specially designed UV lamp in turn for SP-PT-CVG to generate Hg0, which was swept to an AFS detector for its detection. It is worth noting that the tube served as both a vapor generator and a desorption chamber. This proof-of-concept study confirms the feasibility of solid phase CVG. Compared to conventional CVG carried out in the liquid phase, solid phase CVG not only retains the advantages of conventional CVG but also alleviates the matrix interference on vapor generation and preconcentrates analyte prior to vapor generation, improving analytical performance for liquid state samples. DI-SPME-SP-PT-CVG-AFS provides a limit of detection of 2.3 ng L-1 for mercury determination by AFS. In the proposed method, the combination of DI-SPME and SP-PT-CVG eliminates the tedious derivatization steps required in conventional headspace SPME, thus minimizing toxic reagent consumption and improving extraction throughput. The practicality of DI-SPME-SP-PT-CVG-AFS was evaluated by analyzing two different certified reference materials and river water samples with good spike recoveries (98-107%).

Effect of Three Different Amino Acids Plus Gentamicin Against Methicillin-Resistant Staphylococcus aureus.

Background: The issue of methicillin-resistant Staphylococcus aureus (MRSA) resistant to many antibiotics and causing serious infectious diseases is a growing healthcare concern. Purpose: In recent years, exogenous administration of metabolites in combination with antibiotics can re-sensitize resistant bacteria to antibiotics; however, their effects vary, and their underlying mechanism of action remains elusive. Methods: We assessed the bactericidal effects of the three amino acids in combination with gentamicin in vitro and in vivo. Subsequently, we explored the role of these amino acids on the metabolomics of MRSA using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we performed the downstream analyses using MetaboAnalyst and Interactive Pathways Explorer. Results: Exogenous threonine showed the best bactericidal efficacy with gentamicin, followed by glycine, wherein serine had no effect. Amino acid treatments mainly up-regulated the metabolites, increased the amino acid abundance, and significantly activated metabolisms; these effects were consistent with the bactericidal efficacy of the three amino acids. Most amino acids participated in the tricarboxylic acid cycle, and threonine supplementation increased the activities of citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, whereas glycine increased activities of citrate synthase and α-ketoglutarate dehydrogenase, and serine did not affect the activities of any of the three key enzymes. We identified 24 biomarkers in the three groups, among which glutamic acid and cysteine showed a gradient decrease and increase, respectively. Subsequent analyses revealed that glutamic acid but not cysteine promoted the bactericidal effect of gentamicin synergistically. Conclusion: Threonine has the best synergistic effect in reversing bacterial resistance compared to glycine and serine. We show that different amino acids combined with an antibiotic mainly affect amino acid metabolism and act via different metabolic regulatory mechanisms, which could help develop effective strategies for tackling MRSA infections.

Gentamicin resistance to Escherichia coli related to fatty acid metabolism based on transcriptome analysis.

Antibiotic overuse and misuse have promoted the emergence and spread of antibiotic-resistant bacteria. Increasing bacterial resistance to antibiotics is a major healthcare problem, necessitating elucidation of antibiotic resistance mechanisms. In this study, we explored the mechanism of gentamicin resistance by comparing the transcriptomes of antibiotic-sensitive and -resistant Escherichia coli. A total of 410 differentially expressed genes were identified, of which 233 (56.83%) were up-regulated and 177 (43.17%) were down-regulated in the resistant strain compared with the sensitive strain. Gene Ontology (GO) analysis classifies differential gene expression into three main categories: biological processes, cellular components, and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the up-regulated genes were enriched in eight metabolic pathways, including fatty acid metabolism, which suggests that fatty acid metabolism may be involved in the development of gentamicin resistance in E. coli. This was demonstrated by measuring the acetyl-CoA carboxylase activity, plays a fundamental role in fatty acid metabolism, was increased in gentamicin-resistant E. coli. Treatment of fatty acid synthesis inhibitor, triclosan, promoted gentamicin-mediated killing efficacy to antibiotic-resistant bacteria. We also found that exogenous addition of oleic acid, which involved in fatty acid metabolism, reduced E. coli sensitivity to gentamicin. Overall, our results provide insight into the molecular mechanism of gentamicin resistance development in E. coli.

Nozzle Electrode Point Discharge-Enhanced Oxidation and Portable Gas Phase Molecular Fluorescence Spectrometry for Sensitive Field Detection of Sulfide.

It is still a challenge to accurately determine dissolved sulfide due to its susceptibility to contamination and loss during transportation, storage, and analysis in the laboratory, thus highlighting the necessity for its sensitive field analysis. Herein, a robust nozzle electrode point discharge (NEPD) enhanced oxidation coupling with chemical vapor generation (CVG) is described for the highly efficient and flameless conversion of sulfide (S2-) to SO2. Subsequently, a portable and low-power consumption gas phase molecular fluorescence spectrometry (GP-MFS) was constructed for the highly selective and sensitive determination of the generated SO2 via detecting its molecular fluorescence excited by a zinc hollow cathode lamp. Under optimal conditions, a limit of detection (LOD) of 0.1 µM was obtained for dissolved sulfide with a relative standard deviation (RSD, n = 11) of 2.6%. The accuracy and practicability of the proposed method were validated by the analyses of two certified reference materials (CRMs) and several river and lake water samples with satisfactory recoveries of 99%-107%. This work confirms that NEPD enhanced oxidation is a low energy consumption yet highly efficient method for the flameless oxidation of hydrogen sulfide and thus is suitable for the easy field detection of dissolved sulfide in environmental water by CVG-GP-MFS.

A high-throughput atomic emission analyzer for simultaneous field detection of dissolved inorganic and organic carbon in seawater and lake water.

Dissolved inorganic carbon (DIC) and dissolved organic carbon (DOC) are two important indicators of global carbon cycle. However, there are no portable analyzers available to simultaneously accomplish high-throughput field detection of them in the same sample. Herein, a simple analyzer comprising a dual-mode reactor to accomplish both chemical vapor generation and headspace sampling, and a miniature point discharge optical emission spectrometer (µPD-OES) was developed for simultaneous and high-throughput detection of DIC and DOC in seawater and lake water. Phosphoric acid and persulfate were successively injected into sample solutions to convert DIC and DOC to CO2 under the conditions of magnetic stirring and UV irradiation, respectively. Subsequently, the generated CO2 was swept into the µPD-OES for quantitation of DIC and DOC via monitoring carbon atomic emission at 193.0 nm. Under optimal conditions, limits of detection for DIC and DOC (as C) were both 0.01 mg L-1 with relative standard deviations (n = 20) better than 5% and sample throughput of 80 samples per hour. Compared to conventional analyzers, the proposed instrument provides the advantages of high throughput, compactness, low energy consumption and eliminates expensive instruments. The accuracy of the system was validated by simultaneous determination of DIC and DOC in various water samples in laboratory and field environments.

Ampicillin-controlled glucose metabolism manipulates the transition from tolerance to resistance in bacteria.

The mechanism(s) of how bacteria acquire tolerance and then resistance to antibiotics remains poorly understood. Here, we show that glucose abundance decreases progressively as ampicillin-sensitive strains acquire resistance to ampicillin. The mechanism involves that ampicillin initiates this event via targeting pts promoter and pyruvate dehydrogenase (PDH) to promote glucose transport and inhibit glycolysis, respectively. Thus, glucose fluxes into pentose phosphate pathway to generate reactive oxygen species (ROS) causing genetic mutations. Meanwhile, PDH activity is gradually restored due to the competitive binding of accumulated pyruvate and ampicillin, which lowers glucose level, and activates cyclic adenosine monophosphate (cAMP)/cAMP receptor protein (CRP) complex. cAMP/CRP negatively regulates glucose transport and ROS but enhances DNA repair, leading to ampicillin resistance. Glucose and Mn2+ delay the acquisition, providing an effective approach to control the resistance. The same effect is also determined in the intracellular pathogen Edwardsiella tarda. Thus, glucose metabolism represents a promising target to stop/delay the transition of tolerance to resistance.

Uracil restores susceptibility of methicillin-resistant Staphylococcus aureus to aminoglycosides through metabolic reprogramming.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has now become a major nosocomial pathogen bacteria and resistant to many antibiotics. Therefore, Development of novel approaches to combat the disease is especially important. The present study aimed to provide a novel approach involving the use of nucleotide-mediated metabolic reprogramming to tackle intractable methicillin-resistant S. aureus (MRSA) infections. Objective: This study aims to explore the bacterial effects and mechanism of uracil and gentamicin in S. aureus. Methods: Antibiotic bactericidal assays was used to determine the synergistic bactericidal effect of uracil and gentamicin. How did uracil regulate bacterial metabolism including the tricarboxylic acid (TCA) cycle by GC-MS-based metabolomics. Next, genes and activity of key enzymes in the TCA cycle, PMF, and intracellular aminoglycosides were measured. Finally, bacterial respiration, reactive oxygen species (ROS), and ATP levels were also assayed in this study. Results: In the present study, we found that uracil could synergize with aminoglycosides to kill MRSA (USA300) by 400-fold. Reprogramming metabolomics displayed uracil reprogrammed bacterial metabolism, especially enhanced the TCA cycle to elevate NADH production and proton motive force, thereby promoting the uptake of antibiotics. Furthermore, uracil increased cellular respiration and ATP production, resulting the generation of ROS. Thus, the combined activity of uracil and antibiotics induced bacterial death. Inhibition of the TCA cycle or ROS production could attenuate bactericidal efficiency. Moreover, uracil exhibited bactericidal activity in cooperation with aminoglycosides against other pathogenic bacteria. In a mouse mode of MRSA infection, the combination of gentamicin and uracil increased the survival rate of infected mice. Conclusion: Our results suggest that uracil enhances the activity of bactericidal antibiotics to kill Gram-positive bacteria by modulating bacterial metabolism.

Quantitative proteomics reveals reduction in central carbon and energy metabolisms contributes to gentamicin resistance in Staphylococcus aureus.

The emergence of antibiotic resistance greatly increases the difficulty of treating bacterial infections. In order to develop effective treatments, the underlying mechanisms of antibiotic resistance must be understood. In this study, Staphylococcus aureus ATCC6538 strain was passaged in medium with and without gentamicin and obtained lab-evolved gentamicin-resistant S. aureus (RGEN) and gentamicin-sensitive S. aureus (SGEN) strains, respectively. Data-Independent Acquisition (DIA)-based proteomics approach was applied to compare the two strains. A total of 1426 proteins were identified, of which 462 were significantly different: 126 were upregulated and 336 were downregulated in RGEN compared to SGEN. Further analysis found that reduced protein biosynthesis was a characteristic feature in RGEN, related to metabolic suppression. The most differentially expressed proteins were involved in metabolic pathways. In RGEN, central carbon metabolism was dysregulated and energy metabolism decreased. After verification, it was found that the levels of NADH, ATP, and reactive oxygen species (ROS) decreased, and superoxide dismutase and catalase activities increased. These findings suggest that inhibition of central carbon and energy metabolic pathways may play an important role in the resistance of S. aureus to gentamicin, and that gentamicin resistance is associated with oxidative stress. Significance: The overuse and misuse of antibiotics have led to bacterial antibiotic resistance, which is a serious threat to human health. Understanding the mechanisms of antibiotic resistance will help better control these antibiotic-resistant pathogens in the future. The present study characterized the differential proteome of gentamicin-resistant Staphylococcus aureus using the most advanced DIA-based proteomics technology. Many of the differential expressed proteins were related to metabolism, specifically, reduced central carbon and energy metabolism. Lower levels of NADH, ROS, and ATP were detected as a consequence of the reduced metabolism. These results reveal that downregulation of protein expression affecting central carbon and energy metabolisms may play an important role in the resistance of S. aureus to gentamicin.