Succinate and inosine coordinate innate immune response to bacterial infection.

Macrophages restrict bacterial infection partly by stimulating phagocytosis and partly by stimulating release of cytokines and complement components. Here, we treat macrophages with LPS and a bacterial pathogen, and demonstrate that expression of cytokine IL-1ß and bacterial phagocytosis increase to a transient peak 8 to 12 h post-treatment, while expression of complement component 3 (C3) continues to rise for 24 h post-treatment. Metabolomic analysis suggests a correlation between the cellular concentrations of succinate and IL-1ß and of inosine and C3. This may involve a regulatory feedback mechanism, whereby succinate stimulates and inosine inhibits HIF-1α through their competitive interactions with prolyl hydroxylase. Furthermore, increased level of inosine in LPS-stimulated macrophages is linked to accumulation of adenosine monophosphate and that exogenous inosine improves the survival of bacterial pathogen-infected mice and tilapia. The implications of these data suggests potential therapeutic tools to prevent, manage or treat bacterial infections.

Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

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.

Reduced ROS-mediated antibiotic resistance and its reverting by glucose in Vibrio alginolyticus.

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.

Differential Antibody Responses to Outer Membrane Proteins Contribute to Differential Immune Protections between Live and Inactivated Vibrio parahemolyticus.

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.

NaCl promotes antibiotic resistance by reducing redox states in Vibrio alginolyticus.

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.

Polyvalent protective immunogens identified from outer membrane proteins of Vibrio parahaemolyticus and their induced innate immune response.

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.

Neuroglobin Regulates Wnt/ß-Catenin and NFκB Signaling Pathway through Dvl1.

Neuroglobin is an endogenous neuroprotective protein, but the underlying neuroprotective mechanisms remain to be elucidated. Our previous yeast two-hybrid screening study identified that Dishevelled-1, a key hub protein of Wnt/ß-Catenin signaling, is an interaction partner of Neuroglobin. In this study, we further examined the role of Neuroglobin in regulating Dishevelled-1 and the downstream Wnt/ß-Catenin and NFκB signaling pathway. We found that Neuroglobin directly interacts with Dishevelled-1 by co-immunoprecipitation, and the two proteins are co-localized in both cytoplasma and nucleus of SK-N-SH cells. Moreover, the ectopic expression of Neuroglobin promotes the degradation of exogenous and endogenous Dishevelled-1 through the proteasomal degradation pathway. Furthermore, our results showed that Neuroglobin significantly inhibits the luciferase activity of Topflash reporter and the expression of ß-Catenin mediated by Dishevelled-1 in SK-N-SH cells. In addition, we also documented that Neuroglobin enhances TNF-α-induced NFκB activation via down-regulating Dishevelled-1. Finally, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays showed that Neuroglobin is an important neuroprotectant that protects SK-N-SH cells from TNF-α-induced decrease in cell viability. Taken together, these findings demonstrated that Neuroglobin functions as an important modulator of the Wnt/ß-Catenin and NFκB signaling pathway through regulating Dishevelled-1.

Metabolic Mechanism for l-Leucine-Induced Metabolome To Eliminate Streptococcus iniae.

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-1ß 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.

TNFAIP1 contributes to the neurotoxicity induced by Aß25-35 in Neuro2a cells.

BACKGROUND: Amyloid-beta (Aß) accumulation is a hallmark of Alzheimer's disease (AD) that can lead to neuronal dysfunction and apoptosis. Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an apoptotic protein that was robustly induced in the transgenic C. elegans AD brains. However, the roles of TNFAIP1 in AD have not been investigated. RESULTS: We found TNFAIP1 protein and mRNA levels were dramatically elevated in primary mouse cortical neurons and Neuro2a (N2a) cells exposed to Aß25-35. Knockdown and overexpression of TNFAIP1 significantly attenuated and exacerbated Aß25-35-induced neurotoxicity in N2a cells, respectively. Further studies showed that TNFAIP1 knockdown significantly blocked Aß25-35-induced cleaved caspase 3, whereas TNFAIP1 overexpression enhanced Aß25-35-induced cleaved caspase 3, suggesting that TNFAIP1 plays an important role in Aß25-35-induced neuronal apoptosis. Moreover, we observed that TNFAIP1 was capable of inhibiting the levels of phosphorylated Akt and CREB, and also anti-apoptotic protein Bcl-2. TNFAIP1 overexpression enhanced the inhibitory effect of Aß25-35 on the levels of p-CREB and Bcl-2, while TNFAIP1 knockdown reversed Aß25-35-induced attenuation in the levels of p-CREB and Bcl-2. CONCLUSION: These results suggested that TNFAIP1 contributes to Aß25-35-induced neurotoxicity by attenuating Akt/CREB signaling pathway, and Bcl-2 expression.

Liver functional metabolomics discloses an action of L-leucine against Streptococcus iniae infection in tilapias.

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.

Live Edwardsiella tarda vaccine enhances innate immunity by metabolic modulation in zebrafish.

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.

GC/MS-based metabolomics approach to identify biomarkers differentiating survivals from death in crucian carps infected by Edwardsiella tarda.

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.

Bcl-2-associated transcription factor 1 interacts with fragile X-related protein 1.

The absence of fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS), which is the leading cause of hereditary mental retardation. Fragile X-related protein 1 (FXR1P), which plays an important role in normal muscle development, is one of the two autosomal paralogs of FMRP. To understand the functions of FXR1P, we screened FXR1P-interacting proteins by using a yeast two-hybrid system. The fragile X-related gene 1 (FXR1) was fused to pGBKT7 and then used as the bait to screen the human fetal brain cDNA library. The screening results revealed 10 FXR1P-interacting proteins including Bcl-2-associated transcription factor 1 (BTF). The interaction between FXR1P and BTF was confirmed by using both ß-galactosidase assay and growth test in selective media. Co-immunoprecipitation assay in mammalian cells was also carried out to confirm the FXR1P/BTF interaction. Moreover, we confirmed that BTF co-localized with FXR1P in the cytoplasm around the nucleus in rat vascular smooth muscle cells by using confocal fluorescence microscopy. These results provide clues to elucidate the relationship between FXR1P and FXS.

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.

Metabolomic Effects of the Dietary Inclusion of Hermetia illucens Larva Meal in Tilapia.

Black soldier fly (Hermetia illucens) larvae meal have been used as feed protein supplements in fish feed, but few researches have investigated the metabolomic effects of Hermetia illucens larvae meal supplements. Therefore, the metabolic effects on Nile tilapia were investigated by replacing 5%, 10%, and 20% of the dietary soybean meal in the basal diet with Hermetia illucens larvae meal, respectively. This study shows that 20% H. illucens larvae meal feed could promote tilapia average daily gain of upto 5.03 ± 0.18 g (mean ± SEM). It was found that the tricarboxylic acid cycle efficiency was improved by activating the enzymes of mitochondrial isocitrate dehydrogenase, NAD-malate dehydrogenase, succinate dehydrogenase, pyruvate dehydrogenase, and α-ketoglutarate dehydrogenase, which then increased the output of ATP and NADH. Furthermore, amino acid and protein biosynthesis was boosted by enhanced glutamine synthetase and glutamate synthase. In particular, GSH increased with increased H. illucens larvae meal. Unsaturated fatty acid biosynthesis was stimulated by higher levels of fatty acid synthase and acetyl CoA carboxylase. Additionally, there was no significant change in lipase levels. Thus, the higher acetyl Co-A content was primarily involved in fatty acid biosynthesis and energy metabolism. Flavor substances, such as nonanal and 2-methyl-3-furanthiol, also accumulated with the addition of H. illucens larvae meal, which increased the umami taste and meat flavor. Additionally, the flavor of tilapia was improved owing to a decrease in trimethylamine content, which causes an earthy and fishy taste. This study uncovers a previously unknown metabolic effect of dietary H. illucens larvae meal on Nile tilapia.

Genome Sequencing of Hericium coralloides by a Combination of PacBio RS II and Next-Generation Sequencing Platforms.

The fruiting bodies or mycelia of Hericium coralloides (H. coralloides) contain many physiologically active compounds that are used to treat various diseases, including cardiovascular disorders and cancers. However, the genome of H. coralloides has not been sequenced, which hinders further investigations into aspects, such as bioactivity or evolutionary events. The present study is aimed at (i) performing de novo sequencing of the assembled genome; (ii) mapping the reads from PE400 DNA into the assembled genome; (iii) identifying the full length of all the repeated sequences; and (iv) annotating protein-coding genes using GO, eggNOG, and KEGG databases. The assembled genome comprised 5,59,05,675 bp, including 307 contigs. The mapping rate of reads obtained from PE400 DNA in the assembled genome was 92.46%. We identified 2,525 repeated sequences of 14,23,274 bp length. We predicted ncRNAs of 48,895 bp and 11,736 genes encoding proteins that were annotated in the GO, eggNOG, and KEGG databases. We are the first to sequence the entire H. coralloides genome (NCBI; Assembly: ASM367540v1), which will serve as a reference for studying the evolutionary diversification of edible and medicinal mushrooms and facilitate the application of bioactivity in H. coralloides.

Myo-Inositol Restores Tilapia's Ability Against Infection by Aeromonas sobria in Higher Water Temperature.

Bacterial infection presents severe challenge to tilapia farming, which is largely influenced by water temperature. However, how water temperature determines tilapias' survival to infection is not well understood. Here, we address this issue from the perspective of metabolic state. Tilapias were more susceptible to Aeromonas sobria infection at 33°C than at 18°C, which is associated with differential metabolism of the fish. Compared to the metabolome of tilapia at 18°C, the metabolome at 33°C was characterized with increased an tricarboxylic acid cycle and a reduced level of myo-inositol which represent the most impactful pathway and crucial biomarker, respectively. These alterations were accompanied with the elevated transcriptional level of 10 innate immune genes with infection time, where il-1b, il-6, il-8, and il-10 exhibited a higher expression at 33°C than at 18°C and was attenuated by exogenous myo-inositol in both groups. Interestingly, exogenous myo-inositol inactivated the elevated TCA cycle via inhibiting the enzymatic activity of succinate dehydrogenase and malate dehydrogenase. Thus, tilapias showed a higher survival ability at 33°C. Our study reveals a previously unknown relationship among water temperature, metabolic state, and innate immunity and establishes a novel approach to eliminate bacterial pathogens in tilapia at higher water temperature.

Serine Metabolism Tunes Immune Responses To Promote Oreochromis niloticus Survival upon Edwardsiella tarda 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.

Metabolomics study reveals the potential evidence of metabolic reprogramming towards the Warburg effect in precancerous lesions.

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.

Glutamine promotes antibiotic uptake to kill multidrug-resistant uropathogenic bacteria.

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 Escherichia coli using a comparative metabolomics approach. Exogenous glutamine promoted ß-lactam­, aminoglycoside-, quinolone-, and tetracycline-induced killing of uropathogenic E. coli and potentiated ampicillin to eliminate multidrug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella peneumoniae, Edwardsiella tarda, Vibrio alginolyticus, and Vibrio parahaemolyticus. Glutamine-potentiated ampicillin-mediated killing was effective against biofilms of these bacteria in a mouse urinary tract infection model and against systemic infection caused by E. coli, P. aeruginosa, A. baumannii, or K. peneumoniae in a mouse model. Exogenous glutamine stimulated influx of ampicillin, leading to the accumulation of intracellular antibiotic concentrations that exceeded the amount tolerated by the multidrug-resistant bacteria. Furthermore, we demonstrated that exogenous glutamine promoted the biosynthesis of nucleosides including inosine, which in turn interacted with CpxA/CpxR and up-regulated OmpF. We validated the physiological relevance of the mechanism by showing that loss of purF, purH, cpxA, or ompF elevated antibiotic resistance in antibiotic-sensitive strains. In addition, glutamine retarded the development of ampicillin resistance. These results may facilitate future development of effective approaches for preventing or managing chronic, multidrug-resistant bacterial infections, bacterial persistence, and difficult-to-treat bacterial biofilms.