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The mechanism(s) of how bacteria acquire tolerance and then resistance to antibiotics remains poorly understood. Here, we show that glucose abundance decreases progressively as ampicillin-sensitive strains acquire resistance to ampicillin. The mechanism involves that ampicillin initiates this event via targeting pts promoter and pyruvate dehydrogenase (PDH) to promote glucose transport and inhibit glycolysis, respectively. Thus, glucose fluxes into pentose phosphate pathway to generate reactive oxygen species (ROS) causing genetic mutations. Meanwhile, PDH activity is gradually restored due to the competitive binding of accumulated pyruvate and ampicillin, which lowers glucose level, and activates cyclic adenosine monophosphate (cAMP)/cAMP receptor protein (CRP) complex. cAMP/CRP negatively regulates glucose transport and ROS but enhances DNA repair, leading to ampicillin resistance. Glucose and Mn 2+ delay the acquisition, providing an effective approach to control the resistance. The same effect is also determined in the intracellular pathogen Edwardsiella tarda. Thus, glucose metabolism represents a promising target to stop/delay the transition of tolerance to resistance.

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.

Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of double-stranded RNA (dsRNA) that activates anti-infective innate immunity. The underlying mechanisms are identified as targeting pattern recognition receptors and Th1-inducing. However, whether poly I:C manipulates metabolism to implement this anti-infective function is unknown. Here, GC-MS based metabolomics was used to characterize metabolic profiles induced by different doses of poly I:C. Analysis on the dose-dependent metabolomes shows that elevation of the TCA cycle and malate with the increasing dose of ploy I:C forms the most characteristic feature of the poly I:C stimulation. Exogenous malate activates the TCA cycle and elevates survival of zebrafish infected with Vibrio alginolyticus, which is related to the elevated expression of il-1b, il-6, il-8, tnf-a, and c3b. These results reveal a previously unknown regulation of poly I:C that boosts the TCA cycle to enhance innate immunity against bacterial infection.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant 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

Introduction: The understanding of novel antibiotic resistance mechanisms is essential to develop strategies against antibiotic-resistant pathogens, which has become an urgent task due to the worldwide emergence of antibiotic resistance. Areas covered: In this review, the authors summarize the recent progress on antibiotic resistance caused by lab-evolved bacteria and clinical multidrug-resistant bacterial pathogens from the proteomics perspective. Expert opinion: Proteomics provides a new platform for a comprehensive understanding of change in protein pathways that are engaged in antibiotics resistance, which is different from a genetic view that focuses on the role of an individual gene or protein. Further work is required to understand why and how the involved pathways are integrated for surviving antibiotic-mediated killing, to use other OMICs for better comprehension of antibiotic resistance mechanisms, and to develop reprogramming proteomics, which reverts an 'antibiotic resistance proteome' to an 'antibiotic sensitive or antibiotic sensitive-like' proteome, for the control of antibiotic-resistant pathogens.

Serum resistance is a poorly understood but common trait of some difficult-to-treat pathogenic strains of bacteria. Here, we report that glycine, serine and threonine catabolic pathway is down-regulated in serum-resistant Escherichia coli, whereas exogenous glycine reverts the serum resistance and effectively potentiates serum to eliminate clinically-relevant bacterial pathogens in vitro and in vivo. We find that exogenous glycine increases the formation of membrane attack complex on bacterial membrane through two previously unrecognized regulations: 1) glycine negatively and positively regulates metabolic flux to purine biosynthesis and Krebs cycle, respectively. 2) α-Ketoglutarate inhibits adenosine triphosphate synthase, which in together promote the formation of cAMP/CRP regulon to increase the expression of complement-binding proteins HtrE, NfrA, and YhcD. The results could lead to effective strategies for managing the infection with serum-resistant bacteria, an especially valuable approach for treating individuals with weak acquired immunity but a normal complement system., Serum-resistant bacteria can escape complement killing in the bloodstream. Here, using metabolomics and metabolite perturbations, the authors describe an altered metabolic state in serum-resistant Escherichia coli and show that exogenous glycine potentiates elimination of pathogenic bacteria in vivo.

Metabolic flexibility of Pseudomonas aeruginosa could lead to new strategies to combat bacterial infection. The present study used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate global metabolism in naturally and artificially evolved strains with cefoperazone-sulbactam (SCF) resistance (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). Inactivation of the pyruvate cycle and nitric oxide (NO) biosynthesis was identified as characteristic features of SCF resistance in both evolved strains. Nitrite-dependent NO biosynthesis instead of an arginine-dependent NO pathway is responsible for the reduced NO, which is attributed to lower nitrite and electrons from the oxidation of NADH to NAD+ provided by the pyruvate cycle. Exogenous fumarate, NADH, nitrate, and nitrite promoted the NO level and thereby potentiated SCF-mediated killing. Unexpectedly, fumarate caused the elevation of nitrite, while nitrite/nitrate resulted in the increase of Cyt bc1 complex (providing electrons). These interesting findings indicate that the nitrite-dependent NO biosynthesis and the pyruvate cycle are mutual to promote NO metabolism. In addition, the NO-potentiated sensitivity to SCF was validated by NO donor sodium nitroprusside. These results reveal an endogenous NO-mediated SCF resistance and develop its reversion by metabolites in P. aeruginosa. IMPORTANCE Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance. Since bacterial metabolic state impacts sensitivity and resistance to antibiotics, exploring and disclosing bacterial metabolic mechanisms can be used to develop a metabolome-reprogramming approach to elevate bacterial sensitivity to antibiotics. Therefore, GC-MS-based metabolomics is used to explore the similarities and differences of metabolomes between naturally and artificially evolved cefoperazone-sulbactam (SCF)-resistant P. aeruginosa (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). It identifies the depressed nitrite-dependent nitric oxide (NO) biosynthesis as the most overlapping characteristic feature between AP-RCLIN-EVO and AP-RLAB-EVO. This is because the pyruvate cycle fluctuates, thereby generating fewer NADH and providing fewer electrons for nitrite-dependent NO biosynthesis than the control. Interestingly, exogenous fumarate, NADH, nitrate, and nitrite as well as NO donor sodium nitroprusside promote NO generation to elevate sensitivity to SCF. These results highlight the way to understand metabolic mechanisms of antibiotic resistance and explore metabolic modulation to combat the bacterial pathogen.

The present study explored the cooperative effect of both alanine (Ala) and gentamicin (Gent) on metabolic mechanisms by which exogenous Ala potentiates Gent to kill antibiotic-resistant Vibrio alginolyticus. To test this, GC-MS-based metabolomics was used to characterize Ala-, Gent- and both-induced metabolic profiles, identifying nitric oxide (NO) production pathway as the most key clue to understand metabolic mechanisms. Gent, Ala and both led to low, lower and lowest activity of total nitric oxide synthase (tNOS) and level of NO, respectively. NOS promoter L-arginine and inhibitor NG-Monomethyl-L-arginine inhibited and promoted the killing, respectively, with the elevation and decrease of NOS activity and NO level. The present study further showed that CysJ is the enzyme-producing NO in V. alginolyticus. These results indicate that the cooperative effect of Ala and Gent causes the lowest NO, which plays a key role in Ala-potentiated Gent-mediated killing.

Background Microbial metabolism confounds antibiotic efficacy. However, information regarding effect of metabolism on cephalosporin antibiotics-mediated killing and Vibrio spp is largely absence, although the drugs are widely used in clinic and the bacteria are pathogens to both human and aquaculture animals. Purpose This study explores the metabolome of cephalosporin antibiotic-resistant Vibrio alginolyticus and analyzes the role of bacterial metabolism in drug and multidrug-resistance. Results The metabolomes of isogenic ceftazidime-resistant V. alginolyticus (VA-RCAZ) and ceftazidime-sensitive V. alginolyticus (VA-S) were analyzed using gas chromatography -mass spectrometry. The metabolome of VA-RCAZ is characterized by inefficient respiration, an inefficient pyruvate cycle (P cycle), increased biosynthesis of fatty acids and decreased membrane proton motive force. This hypothesis was confirmed by the fact that furfural and malonate, inhibitors of pyruvate dehydrogenase and succinate dehydrogenase (P cycle enzymes), respectively, increased resistance of VA-RCAZ to antibiotics, while exposure to triclosan, to inhibit biosynthesis of fatty acids, decreased resistance. Conclusion These results contribute to our understanding of mechanisms of bacterial antibiotic-resistance and may lead to more effective approaches to treat, manage or prevent infections caused by antibiotic-resistant pathogens including those of the Vibrio species.

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 vaccines against more than one species of pathogens are especially important due to the complex ecosystem in aquaculture. We have previously shown that the development of polyvalent vaccines by shuffling six ompA genes from different bacteria with V. parahaemolyticus VP0764 primers. Here, we used the same 6 genes, V. alginolyticus VA0764 and VA1186, V. parahaemolyticus VP0764 and VP1186, E. tarda ompA and E. coli ompA, but with E. tarda ompA primers to develop new polyvalent vaccines. By this approach, we identified 7 potential polyvalent vaccines that were effective against both V. alginolyticus and E. tarda infections. Furthermore, the innate immunity triggered by the vaccines were also explored in three groups, no protection (group I), protection against V. alginolyticus (group II), and protection against both V. alginolyticus and E. tarda (group III). The transcription of IL-1β, IL-6, IL-8, C3b and NF-kB were significantly increased in group II and group III but not group I, where the expression level of group III was higher than group II. In addition, differential activities of succinate dehydrogenase were detected among the three groups. These results indicate the expansion of polyvalent vaccine reservoir with the same shuffling genes but different primers, and promote the understanding of the mechanisms of polyvalent vaccines based on vaccine-induced innate immunity.

Our previous studies demonstrated that molecular breeding via DNA shuffling directs the evolution of polyvalent vaccines with desired traits, which leads to generation of polyvalent ompA vaccines using Vibrio alginolyticus VA0764 primers. Here, we replaced VA0764 primers with Edwardsiella tarda ompA primers to generate new polyvalent ompA vaccines by DNA shuffling of the same five ompA genes from four species of bacteria E. tarda, V. parahaemolyticus, V. alginolyticus and Escherichia coli. We identified four polyvalent vaccine candidates from a eukaryotic expressing library EompAs-FE containing 82 ompAs using active immune protection against V. alginolyticus and E. tarda. Furthermore, we explored mechanisms of polyvalent vaccine candidates by investigation of the innate immune response to these ompAs, and found that expression of IL-1β, IL-8, IL-15, COX-2, IFN-γ, TLR-1, TLR-3 and C3b genes was elevated as a characteristic feature of these polyvalent vaccine candidates. These results indicate that use of different primers to construct a DNA library selects new evolution of polyvalent vaccines with desired traits, and polyvalent ompA vaccines elicit high innate immune response.

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.

Mechanisms by which vaccines enhance immunity to combat bacterial pathogens are not fully understood. Recently, we have found that live Edwardsiella tarda vaccine enhances ability against the bacterial challenge by metabolic modulation in zebrafish. Here we first explored the metabolic modulation promoted by inactivated E. tarda to eliminate the pathogen. Inactivated E. tarda vaccine modulated a similar metabolome to combat with the pathogen in zebrafish as live E. tarda vaccine did. Specifically, both vaccines promoted biosynthesis of unsaturated fatty acids and the TCA cycle. However, due to relatively higher activated TCA cycle in inactivated vaccine than live vaccine, live vaccine promoted higher abundance of palmitate than inactivated vaccine. Consistently, the protection against E. tarda challenge was palmitate dose-dependent. Live vaccine activated higher expression of IL-1β, IL-8,Cox-2 genes and lower expression of IL-15, IL-21 genes than inactivated vaccine, which is similar to the results stimulated by high and low doses of palmitate, respectively. These findings indicate live and inactivated E. tarda vaccines stimulate differential abundances of palmitate that contribute to differential innate immunities against bacterial infection. Thus, metabolic environment contributes to immune response.

We have recently reported that the survival of tilapia, Oreochromis niloticus, during Edwardsiella tarda infection is tightly associated with their metabolome, where the survived O. niloticus has distinct metabolomic profile to dying O. niloticus. Glucose is the key metabolite to distinguish the survival- and dying-metabolome. More importantly, exogenous administration of glucose to the fish greatly enhances their survival for the infection, indicating the functional roles of glucose in metabolome repurposing, known as reprogramming metabolomics. However, the underlying information for the reprogramming is not yet available. Here, GC/MS based metabolomics is used to understand the mechanisms by which how exogenous glucose elevates O. niloticus, anti-infectious ability to E. tarda. Results showed that exogenous glucose promotes stearic acid and palmitic acid biosynthesis but attenuates TCA cycle to potentiate O. niloticus against bacterial infection, which is confirmed by the fact that exogenous stearic acid increases immune protection in O. niloticus against E. tarda infection in a manner of Mx protein. These results indicate that exogenous glucose reprograms O. niloticus anti-infective metabolome that characterizes elevation of stearic acid and palmitic acid and attenuation of the TCA cycle. Therefore, our results proposed a novel mechanism that glucose promotes unsaturated fatty acid biosynthesis to cope with infection, thereby highlighting a potential way of enhancing fish immunity in aquaculture.

Molecular breeding via DNA shuffling directs the evolution of vaccines with desired traits. In the present study, polyvalent OmpA vaccines were generated by DNA shuffling of five ompA genes from four species of bacteria Vibrio parahaemolyticus, V. alginolyticus, Edwardsiella tarda and Escherichia coli. First, a new hybrid OmpA was constructed using VA0764 primers and used for construction of a prokaryotic expressing library PompAs-FV containing 84 ompAs, which were validated by PCR and SDS/PAGE. Then, the 84 ompAs were used to construct a eukaryotic expressing library EompAs-FV for preparing DNA vaccines. Third, extracellular bacterium V. alginolyticus challenge post active immunization using these DNA vaccines was carried out to identify genes with high immunoprotection. Among the 84 ompAs, 17 showed higher or equal immune protection against infection caused by V. alginolyticus than control VA0764. Finally, immune protection against infection caused by intracellular bacterium Edwardsiella tarda was assessed further using the top seven out of the 17 ompAs. This led to identification of three efficient polyvalent vaccines against infections caused by the extracellular bacterium V. alginolyticus and intracellular bacterium E. tarda. In addition, we sequenced genes for understanding mechanisms of the polyvalent vaccines, but association of immune protection with mutation of gene and amino acids is not determined. These results indicate that DNA shuffling is an efficient way to develop polyvalent vaccines against microbial infections.

Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein-protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes.

Streptococcus agalactiae causes severe systemic infections in human and fish. In the present study, we established a pathogen-plasma interaction model by which we explored how S. agalactiae evaded serum-mediated killing. We found that S. agalactiae grew faster in the presence of yellow grouper plasma than in the absence of the plasma, indicating S. agalactiae evolved a way of evading the fish immune system. To determine the events underlying this phenotype, we applied GC-MS-based metabolomics approaches to identify differential metabolomes between S. agalactiae cultured with and without yellow grouper plasma. Through bioinformatics analysis, decreased malic acid and increased adenosine were identified as the most crucial metabolites that distinguish the two groups. Meanwhile, they presented with decreased TCA cycle and elevated purine metabolism, respectively. Finally, exogenous malic acid and adenosine were used to reprogram the plasma-resistant metabolome, leading to elevated and decreased susceptibility to the plasma, respectively. Therefore, our findings reveal for the first time that S. agalactiae utilizes a metabolic trick to respond to plasma killing as a result of serum resistance, which may be reverted or enhanced by exogenous malic acid and adenosine, respectively, suggesting that the metabolic trick can be regulated by metabolites.

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.

Nikaido (1) claims, contrary to our work (2), that the P cycle is not a general mechanism for energy production and that it does not sensitize bacteria toward aminoglycosides. However, the argumentation for both points is not convincing to us. The P cycle is proposed from the stable-isotope–based nontargeted isotope fate detection analysis, in which we found labeled glutamate flux to glucogenesis and identified three additional steps—transforming oxaloacetate (OAA) to citrate through phosphoenolpyruvate (PEP), pyruvate, and acetyl-CoA—to finish the metabolic process. The finding was further supported by 13C-labeled glucose in figure 7G of ref. 2. The PEP-pyruvate-OAA node, also referred to as the anaplerotic node, is a main way … [↵][1]1To whom correspondence may be addressed. Email: pengb26{at}sysu.edu.cn or pxuanx{at}sysu.edu.cn. [1]: #xref-corresp-1-1

// Liping Dai 1 , Xuan-Xian Peng 2 , Eng M. Tan 3 , Jian-Ying Zhang 1 1 Department of Biological Sciences & NIH-Sponsored Border Biomedical Research Center, The University of Texas at El Paso, El Paso, TX 79968, USA 2 School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China 3 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA Correspondence to: Jianying Zhang, e-mail: jzhang@utep.edu Eng M. Tan, e-mail: emtan@scripps.edu Keywords: CAPERα, tumor-associated antigen, hepatocellular carcinoma, cancer biomarkers, tumor angiogenesis Received: November 13, 2015 Accepted: January 31, 2016 Published: February 25, 2016 ABSTRACT Purpose: CAPERα, a tumor-associated antigen, was identified from a cDNA clone with autoantibody from a patient with hepatocellular carcinoma (HCC). It has been implicated, by way of alternative splicing of VEGF pre-mRNA, in the regulation of microvessel formation in Ewing’s sarcoma. In this study, we looked for possible association of alterations in CAPERα with microvessel density in HCC. Methods: Enzyme-linked immunosorbent assay using recombinant CAPERα as antigen were used to detect antibody against CAPERα. Immunohistochemistry (IHC) on liver sections was performed to analyze expression profiles of CAPERα, VEGF and CD34 in HCC and control tissues and was further used to assess the correlation of expression among CAPERα, VEGF and CD34 in HCC development. Results: Autoantibody to CAPERα was highest in HCC (22/76, 28.9%), not detected in prostate cancer (0/79) and at 3.4% (3/88) in breast cancer. In immunohistochemical analysis of grades II and III HCC tissues, significantly decreased immunostaining for CAPERα was observed and this correlated directly with decreased immunostaining for VEGF ( R =0.534, P =0.0003). Using CD34 immunostaining for detecting newly formed microvessels, strong staining was observed in grades II and III HCC. Normal liver sections, all of which have high expression of CAPERα were totally negative for CD34 immunostaining. A significant inverse correlation was seen between CAPERα and CD34 immunostaining ( R =-0.481, P =0.0012). Conclusions: Decreased expression of CAPERα appears to be correlated with appearance of microvessels. It would be of interest to elucidate the cause of altered CAPERα since new formation of microvessels is important in progression of HCC.

A wide variety of bacterial infections is a major challenge in aquaculture. Development of polyvalent vaccines that can fight against as many pathogens as possible is especially necessary. The present study uses DNA shuffling to create a new hybrid OmpA with improved cross-protection against Vibrio alginolyticus and Edwardsiella tarda through the recombination of six OmpA genes from Vibrio parahaemolyticus, V. alginolyticus, E. tarda and Escherichia coli. Out of the 43 recombinant chimeras genes constructed using VA0764 primers, EompAs-19 was demonstrated as an ideal polyvalent vaccine against infections caused V. alginolyticus and E. tarda. Compared with VA0764, OmpAs-19 had three mutations, which may be a molecular basis of EompAs-19 as an efficient polyvalent vaccine against both V. alginolyticus and E. tarda infections. These results develop a polyvalent vaccine that prevents the infections caused by extracellular and intracellular bacteria. Thus, the present study highlights the way to develop polyvalent vaccines against microbial infections by DNA shuffling.

Metabolomics is emerging as a powerful tool for studying metabolic processes, identifying crucial biomarkers responsible for metabolic characteristics and revealing metabolic mechanisms, which construct the content of discovery metabolomics. The crucial biomarkers can be used to reprogram a metabolome, leading to an aimed metabolic strategy to cope with alteration of internal and external environments, naming reprogramming metabolomics here. The striking feature on the similarity of the basic metabolic pathways and components among vastly different species makes the reprogramming metabolomics possible when the engineered metabolites play biological roles in cellular activity as a substrate of enzymes and a regulator to other molecules including proteins. The reprogramming metabolomics approach can be used to clarify metabolic mechanisms of responding to changed internal and external environmental factors and to establish a framework to develop targeted tools for dealing with the changes such as controlling and/or preventing infection with pathogens and enhancing host immunity against pathogens. This review introduces the current state and trends of discovery metabolomics and reprogramming metabolomics and highlights the importance of reprogramming metabolomics.

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.