Abstract
Although cellular metabolic states have been shown to modulate bacterial susceptibility to antibiotics, the interaction between glutamate (Glu) and chloramphenicol (CAP) resistance remains unclear because of the specificity of antibiotics and bacteria. We found that the level of Glu was upregulated in the CAP-resistant strain of Edwardsiella tarda according to a comparative metabolomics approach based on LC–MS/MS. Furthermore, we verified that exogenous metabolites related to Glu, the tricarboxylic acid (TCA) cycle, and glutathione (GSH) metabolism could promote CAP resistance in survival assays. If GSH metabolism or the TCA cycle is inhibited by L-buthionine sulfoximine or propanedioic acid, the promotion of CAP resistance by Glu in the corresponding pathway disappears. According to metabolomic analysis, exogenous Glu could change pantothenate metabolism, affecting GSH biosynthesis and the TCA cycle. These results showed that the glutamate-pantothenate pathway could promote CAP resistance by being involved in the synthesis of GSH, entering the TCA cycle by direct deamination, or indirectly affecting the metabolism of the two pathways by pantothenate. These results extend our knowledge of the effect of Glu on antibiotic resistance and suggest that the potential effect, which may aggravate antibiotic resistance, should be considered before Glu and GSH administration in the clinic.
1. Introduction
Antibiotic resistance is currently one of the most serious threats to public health (). The concern about rising levels of antibiotic resistance is accompanied by the fact that very few new antibiotics are in development, and resistance to new antibiotics appears much quicker than expected. Various resistance mechanisms have been identified, including mutations in drug targets, increased expression of efflux pumps, and the synthesis of enzymes that inactivate antibiotics (). Although most antibiotic resistance studies have focused on genomic mutations and transcriptional regulation, the increased availability of high-throughput technologies has enabled the analysis of intracellular metabolites of bacterial responses to antibiotic stress (; ; ). Several studies have revealed that alterations in cellular metabolic states play an important role in modulating bacterial susceptibility to antibiotics (; ; ).
Metabolic perturbations alter antibiotic lethality through several processes, such as central carbon metabolism, cellular respiration, and purine metabolism (; ; ). The tricarboxylic acid (TCA) cycle and oxidative phosphorylation are central metabolic processes directly disturbing the redox state via reactive oxygen species (ROS) byproducts (). However, ROS have shown completely different characteristics for different bacteria and antibiotics, including enhanced antibiotic lethality, promoted resistance, or irrelevant effects (; ; ; ; ). To eliminate the negative effects of ROS, organisms use glutathione (GSH), an important antioxidant, to maintain reduction potential in the cytoplasm. In eukaryotic cells and gram-negative bacteria, GSH is composed of three amino acids: L-glutamate (Glu), L-cysteine, and glycine (), and has been reported to affect bacterial sensitivity to antibiotics. Similar to the multiple characteristics of ROS, GSH exhibits antibacterial activity synergized with aztreonam, carbenicillin, ceftazidime, and meropenem against Acinetobacter baumannii, Pseudomonas aeruginosa, and E. coli (; ; ) and antagonizes the ability to promote fluoroquinolone resistance in E. coli (). Furthermore, metabolites related to the TCA cycle, such as α-ketoglutarate, succinate, malate, and NADH, can enhance CAP resistance (). These studies demonstrate that the influences of the TCA cycle and redox state are complex and that these compounds disturb bacterial survival through various processes that are not yet fully understood.
In this study, we aimed to explore how the glutamate-related pathway affected the bactericidal activity of CAP based on metabolomic analysis and a survival assay with the exogenous metabolites, as well as to investigate the relationship between Glu and the TCA cycle, GSH metabolism, and pantothenate under CAP stress. We found that glutamate and GSH pathway-related metabolites could promote resistance to several antibiotics; therefore, glutamate and GSH administration should be conducted more cautiously in conditions of bacterial infection.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Edwardsiella tarda ATCC 15947 was obtained from the China Center of Industrial Culture Collection. The CAP-resistant strain (Et-Re) was derived from a CAP-susceptible strain of E. tarda ATCC 15947, as described previously (). Briefly, the original strain of E. tarda ATCC 15947 (Et-Ori) was cultivated in Tryptic Soy Broth (TSB) medium (Solarbio Science & Technology Co., Ltd., Beijing, China) with increased concentrations of CAP (Solarbio Science & Technology Co., Ltd., Beijing, China) at 37°C for 50 passages. After subculturing, the bacteria were transferred onto a TSB agar plate at 37°C overnight, and colonies were randomly selected to measure the minimum inhibitory concentration (MIC). We selected the colony with the highest MIC value as the antibiotic-resistant strain. Both the original and CAP-resistant strains were identified by 16S RNA sequencing.
2.2. Metabolomic analysis
The procedures for sample preparation, metabolomics data acquisition, and analysis were derived from previous metabolomics information (). Briefly, equivalent cells were quenched with 60% (v/v) cold methanol (Sigma) at −40°C for 5 s and collected by centrifugation at 1000 ×g at 4°C for 10 min. After being resuspended by 0.1 mL of pre-cooled methanol at −20°C and incubation for 60 min, the samples were collected by centrifugation at 14,000 ×g for 15 min, and the supernatants were transferred into a fresh microcentrifuge tube and dried in a centrifugal evaporator. The dried metabolite pellets were re-dissolved in 80% methanol and analyzed using LC–MS/MS.
LC–MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher), and the parameters were set up as previous study before (). The raw data of MS files were processed with the Compound Discoverer software (version 3.0, Thermo Fisher Scientific). After removed the background ions and normalized by QC samples, the identification and quantitative results of peaks were used for subsequent statistical analysis. In this study, metabolomics data related to Glu pathway was reanalysis. The metabolites with p-value <0.05 and log2|(FoldChange)| > 1 were defined as differential metabolites. Glu pathway was annotated by the KEGG database1 and Pathview (; ), and the scatter diagram was drawn by the relative content of metabolites normalized with the original strain.
2.3. Antibiotic bactericidal assays
Bacterial cells were cultured in TSB medium overnight at 37°C and collected by centrifugation at 5000 ×g for 5 min. The collected cells were washed three times with sterile saline and diluted to 106 CFU/mL with M9 minimal media containing 1 mM MgSO4 and 0.1 mM CaCl2. The metabolites and/or CAP were added to the medium and incubated at 37°C for 5 h. The concentrations of CAP for Et-Ori and Et-Re were 100 mg/L and 2000 mg/L, respectively, while the concentration of the metabolites was 10 mM. Finally, 10 μL of the culture was removed, diluted, plated on TSB agar plates, and incubated at 37°C for 18 h. The colonies were counted, and the CFU/mL was calculated.
2.4. Metabolome analysis of exogenous addition of Glu
Bacterial cells cultured overnight in TSB medium were harvested, washed with sterile saline, and diluted with M9 minimal medium as described above. Exogenous Glu was added to M9 medium at a concentration of 10 mM and incubated at 37°C for 5 h. The bacterial cells were harvested rapidly by centrifugation at 5000 ×g for 5 min. After washing twice with cold, sterile saline, the bacterial cells were frozen in liquid nitrogen and stored at −80°C. Metabolomic analysis was performed as described previously.
2.5. Survival assay of four antibiotics
Bacterial culture conditions were the same as those used for the survival assay. Briefly, the culture was incubated overnight and diluted to 5 × 105 CFUs/mL. The concentrations of kanamycin (KAN), ceftazidime (CAZ), ciprofloxacin (CIP), and meropenem (MEM) were 10 mg/L, 0.2 mg/L, 3 μg/L, and 0.2 mg/L, respectively. The concentrations of Glu and GSH were 10 mM. Bacteria with Glu/GSH and antibiotics were cultured in a 96-well plate, and the control was cultured with the corresponding antibiotic only. After incubation for 5 h, cell densities were determined using an optical density of 600 nm (OD600). The viability fold was calculated by dividing the OD600 of the experimental group by the control.
3. Results
3.1. Analysis of different metabolites and KEGG pathway of CAP resistance strain
In previous studies, we compared metabolites of Et-Re with Et-Ori via high-performance LC–MS/MS, and 364 different metabolites and 12 different pathways were identified (). In this study, we focused on different metabolites related to the Glu and GSH pathway, including Glu, GSH, glutathione disulfide (GSSG), cysteine (Cys), glycine (Gly), threonine (Thr), methionine (Met), adenosylmethionine (AdoMet), and spermidine (Figure 1A). The relative contents of Glu, Cys, Gly, and Thr increased in Et-Re, while those of the remaining five metabolites decreased. In the KEGG enrichment pathway of GSH metabolism, the increased metabolites of Glu, Gly, and Cys are shown in the red cycle, and the decreased metabolites of GSH, GSSG, and spermidine are shown in the green cycle (Figure 1B).
Figure 1
3.2. Metabolites related to the Glu and GSH pathways promote CAP resistance
The percent survival of Et-Ori and Et-Re depended on the concentrations of Glu and GSH under CAP pressure in the M9 medium. As the concentration of Glu increased gradually, the viabilities of the two groups increased, with a peak concentration of 50 mM in Et-Ori and 25 mM in Et-Re (Figures 2A,B). However, the percent survival declined rapidly when the concentration of Et-Re was more than 50 mM. GSH gradually increased the viability of Et-Ori and Et-Re as the concentration increased, and both had a peak concentration of 50 mM (Figures 2C,D).
Figure 2
CAP resistance could be promoted by the other four metabolites related to the Glu and GSH pathway, including Cys, Thr, Met, and GSSG (Figures 2E,F). The highest percent survival in Et-Ori was in the Cys group, a 14.2-fold increase compared with the control. In Et-Re, Cys and GSSG promoted CAP resistance with 14.8- and 128.2-fold survival percentages, respectively.
3.3. GSH metabolism is important in CAP resistance
GSH is an important intracellular reductant; therefore, we used thiourea (ThR) to verify whether reductants promote CAP resistance. Similar to GSH, which could increase the survival rate by 90.0 folds, ThR dramatically promoted CAP resistance in the M9 medium with a 69.0-fold increase in Et-Ori (Figure 3A). Furthermore, the viabilities of Et-Re were substantially improved by GSH and ThR, with 1,458- and 1,483-fold increases, respectively (Figure 3B). L-Buthionine sulfoximine (BSO) is an inhibitor of glutamylcysteine synthetase, which is involved in glutathione synthesis. With increasing concentrations of BSO, the viabilities gradually decreased in Et-Ori and Et-Re, and Glu could no longer promote CAP resistance (Figures 3C,D).
Figure 3
3.4. Glu affects CAP resistance through the TCA cycle
Glu can be converted to α-ketoglutarate (αKg) and enter the TCA cycle. According to a previous study (), metabolites in the TCA cycle can increase CAP resistance in E. tarda. Glu and the metabolites in the TCA cycle promoted the differences in viability. The percent survival of the Glu group was the highest in the Et-Ori and Et-Re groups (Figures 4A,B). The αKg group had a lower survival rate than the Suc group in Et-Ori, but the opposite was true for Et-Re (Figures 4A,B). Propanedioic acid (PPA) is a competitive inhibitor of succinate dehydrogenase, which could disturb the CAP resistance metabolites provided in the TCA cycle (). Similar to BSO, the survival rates gradually decreased in Et-Ori and Et-Re with increasing concentrations of PPA (Figures 4C,D).
Figure 4
3.5. Analysis of different metabolites and KEGG pathway generated by exogenous glutamate
The metabolites in the exogenous glutamate and control groups were measured by LC–MS/MS. The two groups were completely separated using the PLSDA (Figure 5A). There were 325 metabolites identified, and significantly different metabolites were 144 in the Glu group compared with the control group. There were 72 metabolites with both upregulated and downregulated (Figure 5B). The abundance of metabolites significantly differed between the two groups (Figure 5C). According to KEGG enrichment analysis, there were two significantly different pathways (p < 0.05; Figure 6A): tyrosine metabolism and pantothenate and CoA biosynthesis. The pantothenate pathway has four significantly different metabolites: provitamin B5 (pB5), dephospho-CoA, pantothenate (VB5), and pantetheine. The first two metabolites had upregulated, and the latter two had downregulated (Figures 6B,C). The level of the two metabolites of the TCA cycle, fumarate and succinate, were both downregulated (Figure 6B), but the TCA cycle was not significantly changed.
Figure 5
Figure 6
3.6. Metabolites related to VB5 altered bactericidal ability of CAP
Metabolites in the VB5 pathway have different effects on the bactericidal efficiency of CAP. Provitamin B5 could reduce the viability of E. tarda under the pressure of CAP, but VB5 and β-Ala promote CAP resistance (Figures 7A,B). The percent survival was 0.61-, 3.60-, and 2.80-fold in Et-Ori and 0.36-, 44.9-, and 14.3-fold in Et-Re compared with the control. The effects of these metabolites were more pronounced in Et-Re.
Figure 7
3.7. Glu and GSH promote other antibiotic resistance
To investigate whether Glu and GSH could promote bacterial resistance to other antibiotics, we added Glu or GSH with four antibiotics separately into the TSB medium, including kanamycin (KAN), ceftazidime (CAZ), ciprofloxacin (CIP), and meropenem (MEM). Glu and GSH could increase resistance to the last three antibiotics but only GSH affected MEM (Figures 8A,B). The increase in OD600 of the four antibiotics by Glu was 17.7-, 1.76-, 16.5-, and 1.15-fold (Figure 8A), and the increase in GSH was 12.2-, 2.45-, 23.4-, and 4.09-fold (Figure 8B).
Figure 8
4. Discussion
Mounting evidence has revealed that the cellular metabolic state is important in modulating antibiotic susceptibility in multiple bacterial species (). In our previous study, metabolomic analysis of E. tarda showed that Glu-related metabolism was significantly different in CAP resistance strains (). Subsequently, we found that the percent survival of E. tarda increased as the concentration of Glu increased, demonstrating that Glu could affect the bactericidal ability of CAP. Glutamate is not only an amino acid but is also involved in many important metabolic processes. Glutamate metabolism is closely related to GSH metabolism and the TCA cycle. The synthesis of GSH starts with the combination of Glu and Cys to γ-L-glutamyl-L-cysteine catalyzed by γ-glutamylcysteine synthetase and consumes ATP. In addition, glutamate can be converted to α-ketoglutarate by deamination; therefore, glutamate is a key intermediary between α-amino acids and the TCA cycle.
GSH is an important reductant that protects cells against oxidative stress. Numerous studies have demonstrated that the intracellular redox state is lethal to bacteria under antibiotic stress (; ; ; ). However, the effects of GSH on the bactericidal efficiency of antibiotics have been inconsistent. On the one hand, GSH increases the sensitivity of E. coli and P. aeruginosa to trimethoprim and ciprofloxacin by inhibiting the expression of acrAB-tolC and disrupting biofilms, respectively (; ). On the other hand, GSH inactivates fosfomycin by opening the epoxide ring of the antibiotics (). GSH can protect E. coli against fluoroquinolones (ciprofloxacin, norfloxacin, ofloxacin, and gatifloxacin) by disturbing ROS, but not against nonfluoroquinolones (ampicillin and tetracycline) (). In this study, we found that ThR provided a similar percent survival to GSH, which showed that the reductant could promote CAP resistance. The increased percent survivals of Glu and GSH were positively correlated with their respective concentrations. Metabolites related to GSH metabolism, such as Cys, Met, and GSSG, could promote CAP resistance. Cys is a component of GSH, and Met is the provider of thiol. More importantly, if GSH metabolism is disturbed by the inhibitor of glutamylcysteine synthetase, Glu can no longer promote CAP resistance. As a component of GSH, the effect of Glu on CAP resistance may be achieved by regulating GSH synthesis.
As an important central carbon and energy metabolism pathway, the TCA cycle is associated with antibiotic resistance through its effect on ROS (). It has been demonstrated that perturbation of the TCA cycle could reduce antibiotic sensitivity (), whereas promotion of the TCA cycle restores the susceptibility of bacteria to antibiotics (). Furthermore, lower basal respiration can prevent metabolic toxicity and minimize drug lethality (). In a previous study, we demonstrated that metabolites in the TCA cycle, such as α-ketoglutarate, succinate, fumarate, malate, and oxaloacetate, could increase CAP resistance of E. tarda (). In this study, we found that the percent survival after adding Glu was higher than that of metabolites in the TCA cycle. Furthermore, the increased survival provided by Glu gradually disappeared if the TCA cycle was disturbed by the increased concentration of the competitive inhibitor of succinate dehydrogenase.
Inhibition assays of glutamylcysteine synthetase and succinate dehydrogenase indicated that Glu promoted CAP resistance by directly affecting the synthesis of GSH and the TCA cycle. In addition, the analysis of the exogenous Glu effect on metabolomics showed that Glu could affect CAP resistance via VB5. Pantothenate and CoA biosynthesis was the significantly enriched pathway. Catalyzed by aspartate transaminase, Glu can be converted to aspartate, and the latter resolves into CO2 and β-alanine catalyzed by aspartate decarboxylase to enter pantothenate metabolism. β-Alanine is indispensable for synthesizing pantothenate, as it is a precursor of coenzyme A (CoA), which participates in converting pyruvate to acetyl-CoA for the TCA cycle. Both β-alanine and pantothenate promoted CAP resistance, verifying that the pantothenate pathway played an important role in CAP resistance. On the one hand, Pantothenol (pB5) is a provitamin of pantothenate, an inhibitor of the phosphorylation activity of the prokaryotic pantothenate kinase, which catalyzes the first step of the coenzyme A biosynthetic pathway (). Pantothenol decreased the percent survival rate, which showed that sensitivity to CAP increased when the pantothenate pathway was blocked. On the other hand, pantothenate is an effective modulator of the thiol-disulfide system, which increases the antioxidant capacity and stimulates glutathione biosynthesis (; ). Furthermore, the level of the two metabolites in the TCA cycle, fumarate and succinate, were downregulated due to the influence of exogenous Glu. These two metabolites are located between αKg and oxaloacetate. The former is the node where Glu enters the TCA cycle and the latter is the end of the TCA cycle, which reacts with acetyl-CoA to restart the TCA cycle. The two metabolites were influenced by Glu and pantothenate on the TCA cycle. These results verify that the glutamate-pantothenate pathway plays an important role in CAP resistance by affecting the TCA cycle and GSH metabolism.
Based on the above findings, we found that Glu promotes antibiotic resistance in two ways. First, Glu is directly involved in synthesizing GSH as a raw material or enters the TCA cycle by deamination. Second, Glu can change pantothenate metabolism, which affects glutathione biosynthesis and the TCA cycle. Metabolites related to the TCA cycle could directly increase the CAP resistance of E. tarda (α-ketoglutarate, succinate, fumarate, malate, and oxaloacetate) or be activated as cofactors (thiamine, nicotinate, NADH, and NADPH) based on our previous study (). NADH, primarily derived from the TCA cycle, is a hydrogen provider of NADPH (), which contributes to maintaining the reduction state of GSH (). The effect of Glu on CAP resistance is illustrated in Figure 9. Glutamate-pantothenate metabolism is located in the center of the pathway, and Glu is a communication bridge between GSH and the TCA cycle.
Figure 9
As the therapeutic effect of antibiotics is weakened by drug resistance, an effective adjuvant is required to restore the bactericidal ability of antibiotics. Fumarate has been approved for treating Pseudomonas infection by the U.S. Food and Drug Administration. There are dual characteristics of metabolites in the TCA cycle and GSH metabolism: restoration of susceptibility (; ) and increased viability (). In particular, Glu and GSH can impair the bactericidal ability of multiple antibiotics, including CAP, KAN, CAZ, CIP, and MEM. Although GSH is suggested to inhibit the growth of S. aureus, E. coli, Klebsiella pneumonia, and P. aeruginosa (), metabolite adjuvants must be limited strictly to specific antibiotics and bacteria. Survival of the bacterial population provides more opportunities for rare mutations and the evolution of resistance during antibiotic therapy (; ).
Herein, we found that the glutamate-pantothenate pathway promotes antibiotic resistance by affecting GSH metabolism and the TCA cycle. Our study fills some knowledge gaps in the relationship between Glu, VB5, GSH metabolism, and the TCA cycle. However, antibiotic-mediated cell death is a complex process that varies with different antibiotics and bacteria, leading to the requirement of further research to improve our understanding of the interplay between antibiotics and metabolites.
Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was financially supported by Shandong Provincial Natural Science Foundation of China (ZR2022MC170, ZR2019PH019), Agricultural Seed Project of Shandong Province (2019LZGC013), and Innovation Project of Science and Technology of Shandong Province (2021CXGC010806).
Publisher’s note
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Statements
Data availability statement
The data presented in the study are deposited in the figshare repository, Accession number 10.6084/m9.figshare.24031929.
Author contributions
B-bY: Investigation, Writing – original draft. X-sD: Writing – review & editing, Investigation, Methodology. X-yL: Writing – review & editing, Investigation. LA: Writing – review & editing, Investigation. X-rW: Writing – review & editing, Investigation. L-gZ: Writing – review & editing, Investigation. Q-lM: Conceptualization, Writing – review & editing. CW: Conceptualization, Supervision, Writing – original draft, Writing – review & editing. J-pW: Investigation, Writing - review & editing.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Footnotes
References
1
AlharbeR.AlmansourA.KwonD. H. (2017). Antibacterial activity of exogenous glutathione and its synergism on antibiotics sensitize carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii. Int. J. Med. Microbiol.307, 409–414. doi: 10.1016/j.ijmm.2017.07.009
2
AllisonK. R.BrynildsenM. P.CollinsJ. J. (2011). Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature473, 216–220. doi: 10.1038/nature10069
3
AllocatiN.FedericiL.MasulliM.Di IlioC. (2009). Glutathione transferases in bacteria. FEBS J.276, 58–75. doi: 10.1111/j.1742-4658.2008.06743.x
4
ChohnanS.MuraseM.KurikawaK.HigashiK.OgataY. (2014). Antimicrobial activity of pantothenol against staphylococci possessing a prokaryotic type II pantothenate kinase. Microbes Environ.29, 224–226. doi: 10.1264/jsme2.me13178
5
CohenN. R.LobritzM. A.CollinsJ. J. (2013). Microbial persistence and the road to drug resistance. Cell Host Microbe13, 632–642. doi: 10.1016/j.chom.2013.05.009
6
DaviesB. W.KohanskiM. A.SimmonsL. A.WinklerJ. A.CollinsJ. J.WalkerG. C. (2009). Hydroxyurea induces hydroxyl radical-mediated cell death in Escherichia coli. Mol. Cell36, 845–860. doi: 10.1016/j.molcel.2009.11.024
7
DeponteM. (2013). Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes. Biochim. Biophys. Acta1830, 3217–3266. doi: 10.1016/j.bbagen.2012.09.018
8
DuC. C.YangM. J.LiM. Y.YangJ.PengB.LiH.et al. (2017). Metabolic mechanism for l-leucine-induced metabolome to eliminate Streptococcus iniae. J. Proteome Res.16, 1880–1889. doi: 10.1021/acs.jproteome.6b00944
9
FotiJ. J.DevadossB.WinklerJ. A.CollinsJ. J.WalkerG. C. (2012). Oxidation of the guanine nucleotide pool underlies cell death by bactericidal antibiotics. Science336, 315–319. doi: 10.1126/science.1219192
10
GoswamiM.JawaliN. (2007). Glutathione-mediated augmentation of beta-lactam antibacterial activity against Escherichia coli. J. Antimicrob. Chemother.60, 184–185. doi: 10.1093/jac/dkm121
11
GoswamiM.MangoliS. H.JawaliN. (2006). Involvement of reactive oxygen species in the action of ciprofloxacin against Escherichia coli. Antimicrob. Agents Chemother.50, 949–954. doi: 10.1128/AAC.50.3.949-954.2006
12
KhareA. (2021). Achilles' heel of antibiotic resistance. Nat. Microbiol.6, 1339–1340. doi: 10.1038/s41564-021-00985-x
13
KlareW.DasT.IbugoA.BuckleE.ManefieldM.ManosJ. (2016). Glutathione-disrupted biofilms of clinical Pseudomonas aeruginosa strains exhibit an enhanced antibiotic effect and a novel biofilm transcriptome. Antimicrob. Agents Chemother.60, 4539–4551. doi: 10.1128/AAC.02919-15
14
KohanskiM. A.DePristoM. A.CollinsJ. J. (2010). Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis. Mol. Cell37, 311–320. doi: 10.1016/j.molcel.2010.01.003
15
KohanskiM. A.DwyerD. J.WierzbowskiJ.CottarelG.CollinsJ. J. (2008). Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. Cells135, 679–690. doi: 10.1016/j.cell.2008.09.038
16
Levin-ReismanI.RoninI.GefenO.BranissI.ShoreshN.BalabanN. Q. (2017). Antibiotic tolerance facilitates the evolution of resistance. Science355, 826–830. doi: 10.1126/science.aaj2191
17
LiL.SuY. B.PengB.PengX. X.LiH. (2020). Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus. Environ. Microbiol.22, 4295–4313. doi: 10.1111/1462-2920.15021
18
LopatkinA. J.BeningS. C.MansonA. L.StokesJ. M.KohanskiM. A.BadranA. H.et al. (2021). Clinically relevant mutations in core metabolic genes confer antibiotic resistance. Science371:eaba0862. doi: 10.1126/science.aba0862
19
LuoW.BrouwerC. (2013). Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics29, 1830–1831. doi: 10.1093/bioinformatics/btt285
20
LuoW.PantG.BhavnasiY. K.BlanchardS. G.Jr.BrouwerC. (2017). Pathview web: user friendly pathway visualization and data integration. Nucleic Acids Res.45, W501–W508. doi: 10.1093/nar/gkx372
21
MeylanS.PorterC. B. M.YangJ. H.BelenkyP.GutierrezA.LobritzM. A.et al. (2017). Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control. Cell Chem Biol24, 195–206. doi: 10.1016/j.chembiol.2016.12.015
22
MoiseenokA. G.KomarV. I.KhomichT. I.KanunnikovaN. P.SlyshenkovV. S. (2000). Pantothenic acid in maintaining thiol and immune homeostasis. Biofactors11, 53–55. doi: 10.1002/biof.5520110115
23
PengB.LiH.PengX. X. (2015a). Functional metabolomics: from biomarker discovery to metabolome reprogramming. Protein Cell6, 628–637. doi: 10.1007/s13238-015-0185-x
24
PengB.SuY. B.LiH.HanY.GuoC.TianY. M.et al. (2015b). Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria. Cell Metab.21, 249–262. doi: 10.1016/j.cmet.2015.01.008
25
RicciV.LomanN.PallenM.IvensA.FookesM.LangridgeG. C.et al. (2012). The TCA cycle is not required for selection or survival of multidrug-resistant salmonella. J. Antimicrob. Chemother.67, 589–599. doi: 10.1093/jac/dkr515
26
RosatoR. R.FernandezR.PazL. I.SinghC. R.RosatoA. E. (2014). TCA cycle-mediated generation of ROS is a key mediator for HeR-MRSA survival under β-lactam antibiotic exposure. PLoS One9:e99605. doi: 10.1371/journal.pone.0099605
27
SchairerD. O.ChouakeJ. S.KutnerA. J.MakdisiJ.NosanchukJ. D.FriedmanA. J. (2013). Evaluation of the antibiotic properties of glutathione. J. Drugs Dermatol.12, 1272–1277. doi: 10.1016/j.jaad.2014.01.131
28
SlyshenkovV. S.DymkowskaD.WojtczakL. (2004). Pantothenic acid and pantothenol increase biosynthesis of glutathione by boosting cell energetics. FEBS Lett.569, 169–172. doi: 10.1016/j.febslet.2004.05.044
29
SmirnovaG. V.OktyabrskyO. N. (2005). Glutathione in bacteria. Biochemistry (Mosc)70, 1199–1211. doi: 10.1007/s10541-005-0248-3
30
SongY.ZhouZ.GuJ.YangJ.DengJ. (2021). Reducing the periplasmic glutathione content makes Escherichia coli resistant to trimethoprim and other antimicrobial drugs. Microbiol Spectr9:e0074321. doi: 10.1128/Spectrum.00743-21
31
SuY. B.PengB.HanY.LiH.PengX. X. (2015). Fructose restores susceptibility of multidrug-resistant Edwardsiella tarda to kanamycin. J. Proteome Res.14, 1612–1620. doi: 10.1021/pr501285f
32
SuY. B.PengB.LiH.ChengZ. X.ZhangT. T.ZhuJ. X.et al. (2018). Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria. Proc. Natl. Acad. Sci. U. S. A.115, E1578–E1587. doi: 10.1073/pnas.1714645115
33
ThomasV. C.KinkeadL. C.JanssenA.SchaefferC. R.WoodsK. M.LindgrenJ. K.et al. (2013). A dysfunctional tricarboxylic acid cycle enhances fitness of Staphylococcus epidermidis during β-lactam stress. MBio4:e00437. doi: 10.1128/mBio.00437-13
34
Van AckerH.Van DijckP.CoenyeT. (2014). Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms. Trends Microbiol.22, 326–333. doi: 10.1016/j.tim.2014.02.001
35
VilchezeC.HartmanT.WeinrickB.JacobsW. R.Jr. (2013). Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction. Nat. Commun.4:1881. doi: 10.1038/ncomms2898
36
WalshC. (2000). Molecular mechanisms that confer antibacterial drug resistance. Nature406, 775–781. doi: 10.1038/35021219
37
WangC.DongX. S.YangY. Y.XuG. J.WuM. M.YanF. J.et al. (2021). Metabolites in the TCA cycle promote resistance to chloramphenicol of Edwardsiella tarda. J. Proteome Res.20, 972–981. doi: 10.1021/acs.jproteome.0c00725
38
YeJ. Z.SuY. B.LinX. M.LaiS. S.LiW. X.AliF.et al. (2018). Alanine enhances aminoglycosides-induced ROS production as revealed by proteomic analysis. Front. Microbiol.9:29. doi: 10.3389/fmicb.2018.00029
39
ZhangS.WangJ.JiangM.XuD.PengB.PengX. X.et al. (2019). Reduced redox-dependent mechanism and glucose-mediated reversal in gentamicin-resistant Vibrio alginolyticus. Environ. Microbiol.21, 4724–4739. doi: 10.1111/1462-2920.14811
40
ZhangS.YangM. J.PengB.PengX. X.LiH. (2020). Reduced ROS-mediated antibiotic resistance and its reverting by glucose in Vibrio alginolyticus. Environ. Microbiol.22, 4367–4380. doi: 10.1111/1462-2920.15085
41
ZhaoX. L.ChenZ. G.YangT. C.JiangM.WangJ.ChengZ. X.et al. (2021). Glutamine promotes antibiotic uptake to kill multidrug-resistant uropathogenic bacteria. Sci. Transl. Med.13:eabj0716. doi: 10.1126/scitranslmed.abj0716
Summary
Keywords
antibiotic resistance, Edwardsiella tarda, glutamate, glutathione, chloramphenicol, pantothenate
Citation
Yan B, Dong X, Wang J, Li X, An L, Wang X, Zhang L, Meng Q and Wang C (2023) Glutamate-pantothenate pathway promotes antibiotic resistance of Edwardsiella tarda. Front. Microbiol. 14:1264602. doi: 10.3389/fmicb.2023.1264602
Received
25 July 2023
Accepted
24 August 2023
Published
13 September 2023
Volume
14 - 2023
Edited by
Rustam Aminov, University of Aberdeen, United Kingdom
Reviewed by
Jiong Chen, Ningbo University, China; Volker Behrends, University of Roehampton London, United Kingdom
Updates
Copyright
© 2023 Yan, Dong, Wang, Li, An, Wang, Zhang, Meng and Wang.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Chao Wang, wangchao850303@163.comQing-lei Meng, qingleimeng@126.com
Disclaimer
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