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Antibiotic resistance is growing as a public health concern worldwide. Understanding of antibiotic-resistant mechanisms is especially necessary for control of the antibiotic resistance. The present study determined a proteome of Escherichia coli in response to ampicillin. The proteome consisted of 16-differential abundance of proteins, belonging to 8 pathways and constructing a protein-protein network. iPath analysis showed the reduced central carbon metabolism, oxidative energy production pathways, and fatty acid biosynthesis as the responsible processes. These data are supported by the decreased enzyme activity of the pyruvate cycle, NADH, membrane potential, and ATP. Moreover, the present study identified AdhE and FabG as ampicillin-binding proteins from the 16-differential abundance of proteins. Further study was focused on the mechanism of adhE in the resistance. Ampicillin-stressed E. coli exhibited higher adhE expression with lower intracellular alcohol. However, loss of adhE lowered bacteria viability which was associated with high intracellular alcohol than ampicillin-stressed E. coli. Thus, AdhE plays a role in the resistance through regulation of intracellular alcohol. These findings are helpful in further understanding ampicillin resistance mechanisms.

The prevalence of multidrug-resistant bacteria has been increasing rapidly worldwide, a trend that poses great risk to human and animal health and creates urgent need for pharmaceutical and nonpharmaceutical approaches to stop the spread of disease due to antimicrobial resistance. Here, we found that alanine, aspartate, and glutamate metabolism was inactivated, and glutamine was repressed in multidrug-resistant uropathogenic

Summary Vibrio alginolyticus threatens both humans and marine animals, but hosts respond to V. alginolyticus infection is not fully understood. Here, functional metabolomics was adopted to investigate the metabolic differences between the dying and surviving zebrafish upon V. alginolyticus infection. Tryptophan was identified as the most crucial metabolite, whose abundance was decreased in the dying group but increased in the survival group as compared to control group without infection. Concurrently, the dying zebrafish displayed excessive immune response and produced higher level of reactive oxygen species (ROS). Interestingly, exogenous tryptophan reverted dying rate through metabolome re‐programming, thereby enhancing the survival from V. alginolyticus infection. It is preceded by the following mechanism: tryptophan fluxed into the glycolysis and tricarboxylic acid cycle (TCA cycle), promoted adenosine triphosphate (ATP) production and further increased the generation of NADPH. Meanwhile, tryptophan decreased NADPH oxidation. These together ameliorate ROS, key molecules in excessive immune response. This is further supported by the event that the inhibition of pyruvate metabolism and TCA cycle by inhibitors decreased D. reiro survival. Thus, our data indicate that tryptophan is a key metabolite for the host to fight against V. alginolyticus infection, representing an alternative strategy to treat bacterial infection in an antibiotic‐independent way., The Vibrio alginolyticus infection causes D. reiro death through virulence factors and septic shock‐associated oxidative stress. The oxidative stress could be relived by tryptophan, which fluxes into the TCA cycle to increase the ATP production. ATP served as the substrates for the production of NADPH, which antagonized the ROS, thus protecting host death from over‐active immune response like ROS.

Development of low-cost and eco-friendly approaches to fight bacterial pathogens is especially needed in aquaculture. We previously showed that exogenous malate reprograms zebrafish’s metabolome to potentiate zebrafish survival against Vibrio alginolyticus infection. However, the underlying mechanism is unknown. Here, we use GC-MS based metabolomics to identify the malate-triggered metabolic shift. An activated TCA cycle and elevated taurine are identified as the key metabolic pathways and the most crucial biomarker of the reprogrammed metabolome, respectively. Taurine elevation is attributed to the activated TCA cycle, which is further supported by the increased expression of genes in the metabolic pathway of taurine biosynthesis from the isocitrate of the TCA cycle to taurine. Exogenous taurine increases the survival of zebrafish against V. alginolyticus infection as malate did. Moreover, exogenous taurine and malate regulate the expression of innate immunity genes and promote the generation of reactive oxygen species and nitrogen oxide in a similar way. The two metabolites can alleviate the excessive immune response to bacterial challenge, which protects fish from bacterial infection. These results indicate that malate enhances the survival of zebrafish to V. alginolyticus infection via taurine. Thus, our study highlights a metabolic approach to enhance a host’s ability to fight bacterial infection.

Overactive immune response is a critical factor triggering host death upon bacterial infection. However, the mechanism behind the regulation of excessive immune responses is still largely unknown, and the corresponding control and preventive measures are still to be explored. In this study, we find that Nile tilapia, Oreochromis niloticus, that died from Edwardsiella tarda infection had higher levels of immune responses than those that survived. Such immune responses are strongly associated with metabolism that was altered at 6 h postinfection. By gas chromatography-mass spectrometry-based metabolome profiling, we identify glycine, serine, and threonine metabolism as the top three of the most impacted pathways, which were not properly activated in the fish that died. Serine is one of the crucial biomarkers. Exogenous serine can promote O. niloticus survival both as a prophylactic and therapeutic upon E. tarda infection. Our further analysis revealed exogenous serine flux into the glycine, serine, and threonine metabolism and, more importantly, the glutathione metabolism via glycine. The increased glutathione synthesis could downregulate reactive oxygen species. Therefore, these data together suggest that metabolic modulation of immune responses is a potential preventive strategy to control overactive immune responses. IMPORTANCE Bacterial virulence factors are not the only factors responsible for host death. Overactive immune responses, such as cytokine storm, contribute to tissue injury that results in organ failure and ultimately the death of the host. Despite the recent development of anti-inflammation strategies, the way to tune immune responses to an appropriate level is still lacking. We propose that metabolic modulation is a promising approach in tuning immune responses. We find that the metabolomic shift at as early as 6 h postinfection can be predictive of the consequences of infection. Serine is a crucial biomarker whose administration can promote host survival upon bacterial infection either in a prophylactic or therapeutic way. Further analysis demonstrated that exogenous serine promotes the synthesis of glutathione, which downregulates reactive oxygen species to dampen immune responses. Our study exemplifies that the metabolite(s) is a potential therapeutic reagent for overactive immune response during bacterial infection.

The development of antibiotic resistance in Vibrio alginolyticus represents a threat to human health and fish farming. Environmental NaCl regulation of bacterial physiology is well documented, but whether the regulation contributes to antibiotic resistance remains unknown. To explore this, we compared minimum inhibitory concentration (MIC) of V. alginolyticus cultured in different media with 0.5%-10% NaCl, and found that the MIC increased as the NaCl concentration increased, especially for aminoglycoside antibiotics. Consistent with this finding, internal NaCl also increased, while intracellular gentamicin level decreased. GC-MS-based metabolomics showed different distributions of pyruvate cycle intermediates among 0.5%, 4% and 10% NaCl. Differential activity of enzymes in the pyruvate cycle and altered expression of Na(+)-NQR led to a reducing redox state, characterized by decreased levels of NADH, proton motive force (PMF) and ATP. Meanwhile, NaCl negatively regulated PMF as a consequence of the reducing redox state. These together are responsible for the decreased intracellular gentamicin level with the increased external level of NaCl. Our study reveals a previously unknown redox state-dependent mechanism regulated by NaCl in V. alginolyticus that impacts antibiotic resistance.

Background: Most tumors have an enhanced glycolysis flux, even when oxygen is available, called the aerobic glycolysis or the Warburg effect. Metabolic reprogramming promotes cancer progression, and is even related to the tumorigenesis. However, it is not clear whether the observed metabolic changes act as a driver or a bystander in cancer development. Methods: In this study, the metabolic characteristics of oral precancerous cells and cervical precancerous lesions were analyzed by metabolomics, and the expression of glycolytic enzymes in cervical precancerous lesions was evaluated by RT-PCR and Western blot analysis. Results: In total, 115 and 23 metabolites with reliable signals were identified in oral cells and cervical tissues, respectively. Based on the metabolome, oral precancerous cell DOK could be clearly separated from normal human oral epithelial cells (HOEC) and oral cancer cells. Four critical differential metabolites (pyruvate, glutamine, methionine and lysine) were identified between DOK and HOEC. Metabolic profiles could clearly distinguish cervical precancerous lesions from normal cervical epithelium and cervical cancer. Compared with normal cervical epithelium, the glucose consumption and lactate production increased in cervical precancerous lesions. The expression of glycolytic enzymes LDHA, HK II and PKM2 showed an increased tendency in cervical precancerous lesions compared with normal cervical epithelium. Conclusions: Our findings suggest that cell metabolism may be reprogrammed at the early stage of tumorigenesis, implying the contribution of metabolic reprogramming to the development of tumor.

Antibiotic-resistant Vibrio alginolyticus poses a big challenge to human health and food safety. It is urgently needed to understand the mechanisms underlying antibiotic resistance to develop effective approaches for the control. Here we explored the metabolic difference between gentamicin-resistant V. alginolyticus (VA-RGEN ) and gentamicin-sensitive V. alginolyticus (VA-S), and found that the reactive oxygen species (ROS) generation was altered. Compared with VA-S, the ROS content in VA-RGEN was reduced due to the decreased generation and increased breakdown of ROS. The decreased production of ROS was attributed to the decreased central carbon metabolism, which is associated with the resistance to gentamicin. As such a mechanism, we exogenously administrated VA-RGEN with the glucose that activated the central carbon metabolism and promoted the generation of ROS, but decreased the breakdown of ROS in VA-RGEN . The gentamicin-mediated killing was increased with the elevation of the ROS level by a synergistic effect between gentamicin and exogenous glucose. The synergistic effect was inhibited by thiourea, a scavenger of ROS. These results reveal a reduced ROS-mediated antibiotic resistance mechanism and its reversal by exogenous glucose.

It is widely accepted that live vaccines elicit higher immune protection than inactivated vaccines. However, the mechanisms are largely unknown. Here, an array with 64 recombinant outer membrane proteins of Vibrio parahemolyticus was developed to explore antibody responses of live and inactivated V. parahemolyticus post immunization of the 8th, 12th, 16th and 20th day. Among the 64 outer membrane proteins, 28 elicited antibody generation. They were all detected in live vaccine-induced immunity but only 15 antibodies were found in inactivated vaccine-induced immunity. Passive immunization showed that higher percent survival was detected in live than inactivated vaccine-induced immunities. Active immunization indicated that out of 19 randomly selected outer membrane proteins, 5 stimulated immune protection against V. parahemolyticus infection. Among them, antibodies to VP2309 and VPA0526 were shared in mice immunized by live or inactivated vaccines, whereas antibodies to VPA0548, VPA1745, and VP1667 were only found in mice immunized by live vaccine. In addition, live V. parahemolyticus stimulated earlier antibody response than inactivated bacteria. These results indicate that not all of the outer membrane proteins elicited antibody responses when they work together in the form of live or inactivated bacteria; live vaccine elicits more protective antibodies, which contribute to higher immune protection in live vaccine than inactivated vaccine. Notably, the recombinant proteins might be different from those separated from live bacteria, and they might be different in their immunogenic potencies.

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

Vibrio alginolyticus is a waterborne pathogen that infects a wide variety of hosts including fish and human, and the outbreak of this pathogen can cause a huge economic loss in aquaculture. Thus, enhancing host's capability to survive from V. alginolyticus infection is key to fighting infection and this remains still unexplored. In the present study, we established a V. alginolyticus-zebrafish interaction model by which we explored how zebrafish survived from V. alginolyticus infection. We used GC-MS based metabolomic approaches to characterize differential metabolomes between survival and dying zebrafish upon infection. Pattern recognition analysis identified the TCA cycle as the most impacted pathway. The metabolites in the TCA cycle were decreased in the dying host, whereas the metabolites were increased in the survival host. Furthermore, the enzymatic activities of the TCA cycle including pyruvate dehydrogenase (PDH), α-ketoglutaric dehydrogenase (KGDH) and succinate dehydrogenase (SDH) also supported this conclusion. Among the increased metabolites in the TCA cycle, malic acid was the most crucial biomarker for fish survival. Indeed, exogenous malate promoted zebrafish survival in a dose-dependent manner. The corresponding activities of KGDH and SDH were also increased. These results indicate that the TCA cycle is a key pathway responsible for the survival or death in response to infection caused by V. alginolyticus, and highlight the way on development of metabolic modulation to control the infection.

Vaccines are the most economic, efficient and environment-friendly agents in protecting host against bacterial infection. In aquaculture, polyvalent vaccines targeting more than one bacterial specie are highly demanded due to the presence of various types of bacterial pathogens in farming environment. Here eighteen genes encoding outer membrane proteins of Vibrio parahaemolyticus were cloned and expressed. The expressed recombinant proteins were used for antiserum preparation. Passive and active immune protection of the antiserum and recombinant proteins was investigated in the zebrafish model. Two recombinant proteins, VP1667 and VP2369, showed effective immune protection against at least two genera of bacteria, Vibrio (V. parahaemolyticus and V. alginolyticus), Pseudomonas (P. fluorescens) or/and Aeromonas (A. hydrophila), and thereby are potential polyvalent vaccine candidates to defend against bacterial infection in fish farming. Furthermore, the mechanisms for the two polyvalent vaccines in triggering immune response were explored. Antiserum to VP1667 or VP2369 was not cross-reacted with P. fluorescens and A. hydrophila, whereas both recombinant proteins induced significant innate immune response. Comparatively, VP1667 stimulates stronger lymphokine and monokine, and VP2369 induces stronger humoral immune response, while both produce similar NF-κB, COX-2, TLR-1 and TLR-3 expression. Our results identify two polyvalent vaccines and demonstrate characteristics features of their cross-protection at the content of the innate immune response.

Crucial metabolites that modulate hosts' metabolome to eliminate bacterial pathogens have been documented, but the metabolic mechanisms are largely unknown. The present study explores the metabolic mechanism for l-leucine-induced metabolome to eliminate Streptococcus iniae in tilapia. GC-MS-based metabolomics was used to investigate the tilapia liver metabolic profile in the presence of exogenous l-leucine. Thirty-seven metabolites of differential abundance were determined, and 11 metabolic pathways were enriched. Pattern recognition analysis identified serine and proline as crucial metabolites, which are the two metabolites identified in survived tilapias during S. iniae infection, suggesting that the two metabolites play crucial roles in l-leucine-induced elimination of the pathogen by the host. Exogenous l-serine reduces the mortality of tilapias infected by S. iniae, providing a robust proof supporting the conclusion. Furthermore, exogenous l-serine elevates expression of genes IL-1β and IL-8 in tilapia spleen, but not TNFα, CXCR4 and Mx, suggesting that the metabolite promotes a phagocytosis role of macrophages, which is consistent with the finding that l-leucine promotes macrophages to kill both Gram-positive and Gram-negative bacterial pathogens. Therefore, the ability of phagocytosis enhanced by exogenous l-leucine is partly attributed to elevation of l-serine. These results demonstrate a metabolic mechanism by which exogenous l-leucine modulates tilapias' metabolome to enhance innate immunity and eliminate pathogens.

Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

Control of bacterial infection resides in the core of human health and sustainable animal breeding. Vaccines as an economic and efficient immunoprophylaxis have been widely accepted, but mechanisms for vaccines do not fully understand. Information regarding to metabolome in response to vaccines is not available. Here we explore the metabolic features by using GC/MS based metabolic profile and trace metabolic mechanisms in zebrafish (Dario rerio) in response to live Edwardsiella tarda vaccine. Pathway enrichment analysis shows that live vaccine activates biosynthesis of unsaturated fatty acids and the TCA cycle and reduces aminoacyl-tRNA biosynthesis, suggesting a metabolic characteristic feature in response to the live vaccine. We further demonstrate that hydroxyl radical is limited during stimulation. Finally, we reveal oleate induces effective protection against E. tarda infection. These results have implications for immunity study that metabolic regulation contributes to immune protection. Our findings enable us to propose novel therapeutic strategies on metabolism against bacterial infections.

Streptococcus iniae seriously affects the intensive farming of tilapias. Much work has been conducted on prevention and control of S. iniae infection, but little published information on the metabolic response is available in tilapias against the bacterial infection, and no metabolic modulation way may be adopted to control this disease. The present study used GC/MS based metabolomics to characterize the metabolic profiling of tilapias infected by a lethal dose (LD50) of S. iniae and determined two characteristic metabolomes separately responsible for the survival and dying fishes. A reversal changed metabolite, decreased and increased l-leucine in the dying and survival groups, respectively, was identified as a biomarker which featured the difference between the two metabolomes. More importantly, exogenous l-leucine could be used as a metabolic modulator to elevate survival ability of tilapias infected by S. iniae. These results indicate that tilapias mount metabolic strategies to deal with bacterial infection, which can be regulated by exogenous metabolites such as l-leucine. The present study establishes an alternative way, metabolic modulation, to cope with bacterial infections.

Microbial disease problems constitute the largest single cause of economic losses in aquaculture. An understanding of immune system in aquaculture animals how to function in defense against bacterial infections is especially important to control these diseases and improve food quality and safety. In the present study, we use a crucian carp model to explore which pathways and metabolites are crucial for the defense against infection caused by Edwardsiella tarda EIB202. We establish the metabolic profile of crucian carps and then compare the metabolic difference between survivals and dead fish by self-control. We identify elevating unsaturated fatty acid biosynthesis and decreasing fructose and mannose metabolism as the most key pathways and increasing palmitic acid and decreasing d-mannose as the most crucial metabolites differentiating survivals from death in these fish infected by E. tarda. Our findings highlight the importance of metabolic strategy against bacterial infections.

Article history: Received 19 November 2013 Accepted 29 December 2013 We previously revealed a negative regulation of LamB in chlortetracycline-resistant Escherichia coli strain. In the present study, we first showed that the negative regulation, which was characterized by decreased abundance of LamB with elevated growth of its gene-deleted mutant in medium with antibiotics, was a general response in resistance to different classes of antibiotics using 2-DE based proteomics or/and genetically gene-deletion mutant of LamB. Then, we revealed the interaction of LamB and Odp1 which catalyzes the overall conversion of pyruvate to acetyl-CoA and CO2, and found the decrease of the complex in antibiotic-resistant strains with a minimum inhibitory concentration dose-dependent manner. Further spectrofluorometry assay indicated that LamB served as a porin to influx an antibiotic. Finally, we showed that the decreased expression of LamB and Odp1 was detected in almost of all 34 multidrugresistant strains, which suggested that LamB and Odp1 were biomarkers for identification of antibiotic-resistant E. coli. Our results indicated that the interaction of an outer membrane protein with an energy metabolic enzyme constructed an efficient pathway to resist antibiotics. These findings provide novel insights into the mechanisms of antibiotic resistance. Biological significance Our data indicate that the negative regulation by LamB is widely detected in antibiotic-resistant E. coli. LamB serves as a porin to influx an antibiotic and is interacted with Odp1. The complex decreases in antibiotic-resistant strains with a MIC dose-dependent manner. Our findings indicate that interaction of outer membrane protein with energy metabolic enzyme constructs an efficient pathway to resist antibiotics and provides novel insights into the mechanisms of antibiotic resistance. © 2014 Elsevier B.V. All rights reserved.

Vibrio alginolyticus is the etiological agent that causes great losses in aquacultures and clinical emanating cases in humans. Identification of highly efficient vaccine candidates to control V. alginolyticus infection has been highly concerned since vaccines offer a powerful approach to provide efficient protection from bacterial infections. In the present study, we firstly investigated the altered outer membrane proteins (OM proteins) of V. alginolyticus in response to NaCl concentrations and iron limitation using Western blotting, and then identified the protective activity of these altered OM proteins by bacterial challenge post immunization. Ten OM proteins were differentially expressed in response to the osmolarity changing or/and iron limitation, in which VA2212, OmpV, VPA1186, OmpU, VPA1644, VA1061, VA1631 and VPA0860 were markedly altered in response to osmolarity, and VPA1186, OmpU, OmpV, VA0449, VPA0860, VPA1435 and VA1631 were determined to be iron-limited responsive proteins. Out of the ten OM proteins, VA1061, OmpU, VPA1435 and VPA0860 could be effective vaccine candidates against infection by V. alginolyticus in vivo. Further results indicated that VA1061 and VPA0860 were dominant antigens and could stimulate hosts to produce stronger antibody response than other two in live or inactivated whole-cell vaccines. These results not only expand knowledge on osmolarity-, iron-responsive proteins, but also provide a valuable strategy for identify protective proteins suitable for use in vaccine development.