Functional Proteomics Analysis of Norfloxacin-Resistant Edwardsiella tarda.

Multidrug-resistant Edwardsiella tarda threatens both sustainable aquaculture and human health, but the control measure is still lacking. In this study, we adopted functional proteomics to investigate the molecular mechanism underlying norfloxacin (NOR) resistance in E. tarda. We found that E. tarda had a global proteomic shift upon acquisition of NOR resistance, featured with increased expression of siderophore biosynthesis and Fe3+-hydroxamate transport. Thus, either inhibition of siderophore biosynthesis with salicyl-AMS or treatment with another antibiotic, kitasamycin (Kit), which was uptake through Fe3+-hydroxamate transport, enhanced NOR killing of NOR-resistant E. tarda both in vivo and in vitro. Moreover, the combination of NOR, salicyl-AMS, and Kit had the highest efficacy in promoting the killing effects of NOR than any drug alone. Such synergistic effect not only confirmed in vitro and in vivo bacterial killing assays but also applicable to other clinic E. tarda isolates. Thus, our data suggest a proteomic-based approach to identify potential targets to enhance antibiotic killing and propose an alternative way to control infection of multidrug-resistant E. tarda.

Effect of Moringa oleifera supplementation on productive performance, colostrum composition and serum biochemical indexes of sow.

Moringa oleifera has been considered as a potential functional feed or food, since it contains multiple components beneficial to animal and human. However, little is known about the effects of Moringa oleifera supplementation on productive performances in sows. In the current study, the results showed that dietary Moringa oleifera significantly decreased the farrowing length and the number of stillborn (p < .05), while had an increasing trend in the number of live-born (0.05 < p < .10). Furthermore, 8% Moringa oleifera supplementation significantly elevated protein levels in the colostrum (p < .05); 4% Moringa oleifera lowed serum urea nitrogen of sows after 90 days of gestation (p < .05) and significantly decreased serum glucose on 10 days of lactation (p < .05). Both groups showed significant elevation in serum T-AOC activity (p < .05). The serum malondialdehyde (MDA) of sows declined significantly in 4% Moringa oleifera addition group (p < .05). 8% Moringa oleifera meal significantly elevated serum CAT activity after 60 days of gestation (p < .05), while decreased the serum MDA level and increased the serum GSH-Px activity of sows at 10 days of lactation (p < .05). Of piglets, both two dosages of Moringa oleifera supplementation essentially reduced the serum urea nitrogen (p < .05), and 4% Moringa oleifera meal increased serum total protein (p < .05). In addition, piglets that received 8% Moringa oleifera had the highest serum CAT and SOD activities among all groups (p < .05). The present study indicated that Moringa oleifera supplementation could enhance the reproduction performances, elevate protein levels in the colostrum and improve the serum antioxidant indices in both sows and piglets.

An automatic integrated microfluidic system for allergy microarray chips.

Billions of people suffer from allergies, though in many cases, the source allergen is unknown. If one knows which allergens to avoid, this would result in an improved quality of life. Since a rapid, high-throughput, automatic allergen detection method is of great need, an integrated system combining microfluidic techniques and microarray chips has been developed herein to automate the allergen detection process. The developed microfluidic system could automatically carry out the entire procedure such as reagent incubation, hybridization, transport, and washing without any intermediate step. The microarray chip could be easily detached from the microfluidic chip afterwards, enabling it to be read under a fluorescence scanner. The experimental results indicated that the developed microfluidic system can automatically perform all the incubation processes, including hybridization, reagent transportation, and washing. It is worth noting that active mixing has been applied in the present study which is different from our previous study using micro-channels for passive incubation. Comparable results to a conventional benchtop approach were obtained in ∼30% less time with ∼25% less samples/reagents. Similar results were also demonstrated while detecting immunoglobulin E samples. The developed system could therefore provide a rapid, reliable, and automated approach for detecting allergen-specific antibodies in human serum.

α-Ketoglutarate downregulates thiosulphate metabolism to enhance antibiotic killing.

Potentiation of the effects of currently available antibiotics is urgently required to tackle the rising antibiotics resistance. The pyruvate (P) cycle has been shown to play a critical role in mediating aminoglycoside antibiotic killing, but the mechanism remains unexplored. In this study, we investigated the effects of intermediate metabolites of the P cycle regarding the potentiation of gentamicin. We found that α-ketoglutarate (α-KG) has the best synergy with gentamicin compared to the other metabolites. This synergistic killing effect was more effective with aminoglycosides than other types of antibiotics, and it was effective against various types of bacterial pathogens. Using fish and mouse infection models, we confirmed that the synergistic killing effect occurred in vivo. Furthermore, functional proteomics showed that α-KG downregulated thiosulphate metabolism. Upregulation of thiosulphate metabolism by exogenous thiosulphate counteracted the killing effect of gentamicin. The role of thiosulphate metabolism in antibiotic resistance was further confirmed using thiosulphate reductase knockout mutants. These mutants were more sensitive to gentamicin killing, and less tolerant to antibiotics compared to their parental strain. Thus, our study highlights a strategy for potentiating antibiotic killing by using a metabolite that reduces antibiotic resistance.

Fructose promotes ampicillin killing of antibiotic-resistant Streptococcus agalactiae.

Streptococcus agalactiae (GBS) is an important pathogenic bacteria that infected both aquatic animals and human beings, causing huge economic loss. The increasing cases of antibiotic-resistant GBS impose challenges to treat such infection by antibiotics. Thus, it is highly demanded for the approach to tackle antibiotic resistance in GBS. In this study, we adopt a metabolomic approach to identify the metabolic signature of ampicillin-resistant GBS (AR-GBS) that ampicillin is the routine choice to treat infection by GBS. We find glycolysis is significantly repressed in AR-GBS, and fructose is the crucial biomarker. Exogenous fructose not only reverses ampicillin resistance in AR-GBS but also in clinic isolates including methicillin-resistant Staphylococcus aureus (MRSA) and NDM-1 expressing Escherichia coli. The synergistic effect is confirmed in a zebrafish infection model. Furthermore, we demonstrate that the potentiation by fructose is dependent on glycolysis that enhances ampicillin uptake and the expression of penicillin-binding proteins, the ampicillin target. Our study demonstrates a novel approach to combat antibiotic resistance in GBS.

pts promoter influences antibiotic resistance via proton motive force and ROS in Escherichia coli.

Introduction: Glucose level is related to antibiotic resistance. However, underlying mechanisms are largely unknown. Methods: Since glucose transport is performed by phosphotransferase system (PTS) in bacteria, pts promoter-deleted K12 (Δpts-P) was used as a model to investigate effect of glucose metabolism on antibiotic resistance. Gas chromatography-mass spectrometry based metabolomics was employed to identify a differential metabolome in Δpts-P compared with K12, and with glucose as controls. Results: Δpts-P exhibits the resistance to ß-lactams and aminoglycosides but not to quinolones, tetracyclines, and macrolide antibiotics. Inactivated pyruvate cycle was determined as the most characteristic feature in Δpts-P, which may influence proton motive force (PMF), reactive oxygen species (ROS), and nitric oxide (NO) that are related to antibiotic resistance. Thus, they were regarded as three ways for the following study. Glucose promoted PMF and ß-lactams-, aminoglycosides-, quinolones-mediated killing in K12, which was inhibited by carbonyl cyanide 3-chlorophenylhydrazone. Exogenous glucose did not elevated ROS in K12 and Δpts-P, but the loss of pts promoter reduced ROS by approximately 1/5, which was related to antibiotic resistance. However, NO was neither changed nor related to antibiotic resistance. Discussion: These results reveal that pts promoter regulation confers antibiotic resistance via PMF and ROS in Escherichia coli.

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

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

Functional proteomics identify mannitol metabolism in serum resistance and therapeutic implications in Vibrio alginolyticus.

Serum resistance is recognized as one of the most important pathogenic traits of bacterial pathogens, and no control measure is available. Based on our previous discovery that pathogenic Escherichia coli represses glycine, serine, and threonine metabolism to confer serum resistance and that the reactivation of this pathway by exogenous glycine could restore serum sensitivity, we further investigate the mechanism underlying the action of glycine in Vibrio alginolyticus. Thus, V. alginolyticus is treated with glycine, and the proteomic change is profiled with tandem mass tag-based quantitative proteomics. Compared to the control group, glycine treatment influences the expression of a total of 291 proteins. Among them, a trap-type mannitol/chloroaromatic compound transport system with periplasmic component, encoded by N646_0992, is the most significantly increased protein. In combination with the pathway enrichment analysis showing the altered fructose and mannitol metabolism, mannitol has emerged as a possible metabolite in enhancing the serum killing activity. To demonstrate this, exogenous mannitol reduces bacterial viability. This synergistic effect is further confirmed in a V. alginolyticus-Danio rerio infection model. Furthermore, the mechanism underlying mannitol-enabled serum killing is dependent on glycolysis and the pyruvate cycle that increases the deposition of complement components C3b and C5b-9 on the bacterial surface, whereas inhibiting glycolysis or the pyruvate cycle significantly weakened the synergistic effects and complement deposition. These data together suggest that mannitol is a potent metabolite in reversing the serum resistance of V. alginolyticus and has promising use in aquaculture.

Exogenous glycine promotes oxidation of glutathione and restores sensitivity of bacterial pathogens to serum-induced cell death.

Pathogenic strains of bacteria are often highly adept at evading serum-induced cell death, which is an essential complement-mediated component of the innate immune response. This phenomenon, known as serum-resistance, is poorly understood, and as a result, no effective clinical tools are available to restore serum-sensitivity to pathogenic bacteria. Here, we provide evidence that exogenous glycine reverses defects in glycine, serine and threonine metabolism associated with serum resistance, restores susceptibility to serum-induced cell death, and alters redox balance and glutathione (GSH) metabolism. More specifically, in Vibrio alginolyticus and Escherichia coli, exogenous glycine promotes oxidation of GSH to GSH disulfide (GSSG), disrupts redox balance, increases oxidative stress and reduces membrane integrity, leading to increased binding of complement. Antioxidant or ROS scavenging agents abrogate this effect and agents that generate or potentiate oxidation stimulate serum-mediated cell death. Analysis of several clinical isolates of E. coli demonstrates that glutathione metabolism is repressed in serum-resistant bacteria. These data suggest a novel mechanism underlying serum-resistance in pathogenic bacteria, characterized by an induced shift in the GSH/GSSG ratio impacting redox balance. The results could potentially lead to novel approaches to manage infections caused by serum-resistant bacteria both in aquaculture and human health.

MiR-125b-2 knockout increases high-fat diet-induced fat accumulation and insulin resistance.

Obese individuals are more susceptible to comorbidities than individuals of healthy weight, including cardiovascular disease and metabolic disorders. MicroRNAs are a class of small and noncoding RNAs that are implicated in the regulation of chronic human diseases. We previously reported that miR-125b plays a critical role in adipogenesis in vitro. However, the involvement of miR-125b-2 in fat metabolism in vivo remains unknown. In the present study, miR-125b-2 knockout mice were generated using CRISPR/CAS9 technology, resulting in mice with a 7 bp deletion in the seed sequence of miR-125b-2. MiR-125b-2 knockout increased the weight of liver tissue, epididymal white fat and inguinal white fat. MiR-125b-2 knockout also increased adipocyte volume in HFD-induced obese mice, while there were no significant differences in body weight and feed intake versus mice fed a normal diet. Additionally, qRT-PCR and western blot analysis revealed that the expression of the miR-125b-2 target gene SCD-1 and fat synthesis-associated genes, such as PPARγ and C/EBPα, were significantly up-regulated in miR-125b-2KO mice (P < 0.05). Moreover, miR-125b-2KO altered HFD-induced changes in glucose tolerance and insulin resistance. In conclusion, we show that miR-125b-2 is a novel potential target for regulating fat accumulation, and also a candidate target to develop novel treatment strategies for obesity and diabetes.

Plant MIR167e-5p Inhibits Enterocyte Proliferation by Targeting ß-Catenin.

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. It is worth exploring whether plant miRNAs play a cross-kingdom regulatory role in animals. Herein, we found that plant MIR167e-5p regulates the proliferation of enterocytes in vitro. A porcine jejunum epithelial cell line (IPEC-J2) and a human colon carcinoma cell line (Caco-2) were treated with 0, 10, 20, and 40 pmol of synthetic 2'-O-methylated plant MIR167e-5p, followed by a treatment with 20 pmol of MIR167e-5p for 0, 24, 48, and 72 h. The cells were counted, and IPEC-J2 cell viability was determined by the MTT and EdU assays at different time points. The results showed that MIR167e-5p significantly inhibited the proliferation of enterocytes in a dose- and time-dependent manner. Bioinformatics prediction and a luciferase reporter assay indicated that MIR167e-5p targets ß-catenin. In IPEC-J2 and Caco-2 cells, MIR167e-5p suppressed proliferation by downregulating ß-catenin mRNA and protein levels. MIR167e-5p relieved this inhibition. Similar results were achieved for the ß-catenin downstream target gene c-Myc and the proliferation-associated gene PCNA. This research demonstrates that plant MIR167e-5p can inhibit enterocyte proliferation by targeting the ß-catenin pathway. More importantly, plant miRNAs may be a new class of bioactive molecules for epigenetic regulation in humans and animals.

A nitrocellulose membrane-based integrated microfluidic system for bacterial detection utilizing magnetic-composite membrane microdevices and bacteria-specific aptamers.

Bacteria such as Acinetobacter baumannii (AB) can cause serious infections, resulting in high mortality if not diagnosed early and treated properly; there is consequently a need for rapid and accurate detection of this bacterial species. Therefore, we developed a new, nitrocellulose-based microfluidic system featuring AB-specific aptamers capable of automating the bacterial detection process via the activity of microfluidic devices composed of magnetic-composite membranes. Electromagnets were used to actuate these microfluidic devices such that the entire diagnostic process could be conducted in the integrated microfluidic system within 40 minutes with a limit of detection as low as 450 CFU per reaction for AB. Aptamers were used to capture AB in complex samples on nitrocellulose membranes, and a simple colorimetric assay was used to estimate bacterial loads. Given the ease of use, portability, and sensitivity of this aptamer-based microfluidic system, it may hold great promise for point-of-care diagnostics.