More than Meets the Eye: Untargeted Metabolomics and Lipidomics Reveal Complex Pathways Spurred by Activation of Acid Resistance Mechanisms in Escherichia coli.

Escherichia coli is a ubiquitous group of bacteria that can be either commensal gut microbes or enterohemorrhagic food-borne pathogens. Regardless, both forms must survive acidic environments in the stomach and intestines to reach and colonize the gut, a process that partially relies on amino acid-dependent acid resistance (AR) mechanisms and modifications to membrane phospholipids. However, only the basic tenets of these mechanisms have been elucidated. In this paper, we aim to conduct a full-scale metabolic and lipidomic characterization of E. coli's adaptations to acid stress. We hypothesized that the use of untargeted metabolomics and lipidomics would reveal mechanisms downstream of AR processes that provide novel contributions to acid stress survival. We detected significant differences in the extracellular metabolome and the lipidome induced by amino acid supplementation (glutamine, arginine, or lysine) and contextualized these results using real-time quantitative polymerase chain reaction (RT-qPCR). We additionally identified several metabolic pathways as well as a significant alteration in phospholipid synthetic pathways induced by differential amino acid supplementation. These results demonstrate that AR may extend beyond canonical mechanisms to a coordinated metabolic phenotype. Future studies may benefit from our analysis to further elucidate distinct targets for prebiotic supplements to cultivate commensal strains or therapies to combat pathogenic ones.

Green tea extract alters gut microbiota and their metabolism of adults with metabolic syndrome in a host-free human colonic model.

BACKGROUND: Metabolic syndrome (MetS) is a common metatoblic disorder that leads to various adverse health outcomes such as diabetes and cardiovascular diseases (CVDs). Recent studies suggested that MetS-associated gut dysbiosis could exacerbate MetS related diseases. Green tea, a popular beverage rich in polyphenols, has showed antioxidant and anti-inflammatory effects in treating MetS through gut modulation. OBJECTIVES: This study aimed to understand the impact of green tea extract (GTE) on the composition and metabolism of gut microbiota from people with MetS. METHODS: We utilized an in-vitro human colonic model (HCM) to specifically investigate the host-free interactions between GTE and gut microbiota of MetS adults. Fresh fecal samples donated by three adults with MetS were used as gut microbe inoculum in our HCM system. 16S ribosomal RNA sequencing and liquid-chromatography mass spectrometry (LC/MS) combined with QIIME 2, Compound Discoverer 3.1 and MetaboAnalyst 4.0 based data analyses were performed to show the regulating effects of GTE treatment on gut microbial composition and their metabolism. RESULTS: Our data suggested that GTE treatment in HCM system modified composition of MetS gut microbiota at genus level and led to significant microbiota metabolic profile change. Bioinformatics analysis showed relative abundance of Escherichia and Klebsiella was commonly increased while Bacteroides, Citrobacter, and Clostridium were significantly reduced. All free fatty acids detected were significantly increased in different colon sections. Lipopolysaccharide biosynthesis, methane metabolism, pentose phosphate pathway, purine metabolism, and tyrosine metabolism were regulated by GTE in MetS gut microbiota. In addition, we identified significant associations between altered microbes and microbial metabolites. CONCLUSIONS: Overall, our study revealed the impact of GTE treatment on gut microbiota composition and metabolism changes in MetS microbiota in vitro, which may provide information for further mechanistic investigation of GTE in modulating gut dysbiosis in MetS.

EGCG and catechin relative to green tea extract differentially modulate the gut microbial metabolome and liver metabolome to prevent obesity in mice fed a high-fat diet.

Green tea extract (GTE) alleviates obesity, in part, by modulating gut microbial composition and metabolism. However, direct evidence regarding the catechin-specific bioactivities that are responsible for these benefits remain unclear. The present study therefore investigated dietary supplementation of GTE, epigallocatechin gallate (EGCG), or (+)-catechin (CAT) in male C57BL6/J mice that were fed a high-fat (HF) diet to establish the independent contributions of EGCG and CAT relative to GTE to restore microbial and host metabolism. We hypothesized that EGCG would regulate the gut microbial metabolome and host liver metabolome more similar to GTE than CAT to explain their previously observed differential effects on cardiometabolic health. To test this, we assessed metabolic and phenolic shifts in liver and fecal samples during dietary HF-induced obesity. Ten fecal metabolites and ten liver metabolites (VIP > 2) primarily contributed to the differences in the metabolome among different interventions. In fecal samples, nine metabolic pathways (e.g., tricarboxcylic acid cycle and tyrosine metabolism) were differentially altered between the GTE and CAT interventions, whereas three pathways differed between GTE and EGCG interventions, suggesting differential benefits of GTE and its distinctive bioactive components on gut microbial metabolism. Likewise, hepatic glycolysis / gluconeogenesis metabolic pathways were significantly altered between GTE and EGCG interventions, while only hepatic tyrosine metabolism was altered between CAT and GTE interventions. Thus, our findings support that purified catechins relative to GTE uniquely contribute to regulating host and microbial metabolic pathways such as central energy metabolism to protect against metabolic dysfunction leading to obesity.

Black raspberry extract shifted gut microbe diversity and their metabolic landscape in a human colonic model.

Human gut microbiota is critical for human health, as their dysbiosis could lead to various diseases such as irritable bowel syndrome and obesity. Black raspberry (BRB) has been increasingly studied recently for its impact on gut microbiota as a rich source of phytochemicals (e.g., anthocyanin). To investigate the effect of BRB extract on the gut microbiota composition and their metabolism, an in-vitro human colonic model (HCM) was utilized to study the direct interaction between BRB and gut microbiome. Conditions (e.g., pH, temperature, anaerobic environment) in HCM were closely monitored and maintained to simulate the human intestinal system. Fresh fecal samples donated by three young healthy volunteers were used for gut microbiota inoculation in the HCM. 16S ribosomal DNA sequencing and liquid-chromatography mass spectrometry (LC/MS) based metabolomics were performed to study the impact of BRB on gut microbiota characteristics and their metabolism (fatty acids, polar metabolites, and phenolic compounds). Our data suggested that BRB intervention modulated gut microbiota at the genus level in different HCM sections mimicing ascending, transverse, and descending colons. Relative abundance of Enterococcus was commonly decreased in all colon sections, while modulations of other bacteria genera were mostly location-dependent. Meanwhile, significant changes in the metabolic profile of gut microbiota related to fatty acids, endogenous polar metabolites, and phenolic compounds were detected, in which arginine and proline metabolism, lysine degradation, and aminoacyl-tRNA biosynthesis were mostly regulated. Moreover, we identified several significant associations between altered microbial populations and changes in microbial metabolites. In summary, our study revealed the impact of BRB intervention on gut microbiota composition and metabolism change, which may exert physiological change to host metabolism and host health.

Secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) fingerprinting enabled treatment monitoring of pulmonary carcinoma cells in real time.

Lung cancer is one of the leading causes of cancer related deaths in the United States. A novel volatile analysis platform is needed to complement current diagnostic techniques and better elucidate chemical signatures of lung cancer and subsequent treatments. A systems biology bottom-up approach using cell culture volatilomics was employed to identify pathological volatile fingerprints of lung cancer in real time. An advanced secondary electrospray ionization (SESI) source, named SuperSESI was used in this study and directly attached to a Thermo Q-Exactive high-resolution mass spectrometer (HRMS). We performed a series of experiments to determine if our optimized SESI-HRMS platform can distinguish between cancer types by sampling their in vitro volatilome profiles. We detected 60 significant volatile organic compound (VOC) features in positive mode that were deemed of cancer cell origin. The cell derived features were used for subsequent analyses to distinguish between our two studied lung cancer types, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Partial least squares-discriminant analysis (PLS-DA) model revealed a good separation of the two cancer types, suggesting unique chemical composition of their headspace profiles. A receiver operating characteristic (ROC) curve using 10 prominent features was used to predict disease type, with an area under the curve (AUC) of 0.811. Cultures were also treated with cisplatin to determine the feasibility of classifying drug treatment from expelled gases. A PLS-DA model revealed independent clustering based on their headspace profiles. An ROC curve using the top features driving separation of PLS-DA model suggested good accuracy with an AUC of 1. It is thus possible to benefit from the advantages of this platform to distinguish the unique volatile fingerprints of cancers to uncover potential biomarkers for cancer type differentiation and treatment monitoring.

Monitoring the Diversity and Metabolic Shift of Gut Microbes during Green Tea Feeding in an In Vitro Human Colonic Model.

The human gut microbiome plays an important role in human health, and many factors such as environment, host genetics, age, and diet have been found to influence the microbial composition. Tea, as one of the widely consumed beverages, has been known for centuries to have antioxidant, anti-inflammatory, and anticancer effects. To investigate the impact of green tea polyphenol on the diversity and metabolic functions of human gut microbes, we applied an in vitro human colonic model (HCM) in this study to mimic a short-term green tea ingestion event and investigate its related changes to gut microbial composition and their metabolic functions. The pH, temperature, anaerobic environment, feeding nutrient, and time point in each compartment of the HCM were tightly controlled to simulate the intestinal system, and pooled human fecal samples of two healthy volunteers were used for the colon microbiota inoculation within the colonic model. By adding green tea extract (GTE) to the growth medium, the detailed impacts of GTE polyphenol on gut microbial population/diversity, gut microbial metabolites, metabolic pathways, and their associations were investigated via 16 S ribosomal DNA sequencing and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analyses. Our data indicated that the treatment of green tea extract applied to gut microbiota can induce a significant decrease in the abundance of Firmicutes and a slight decrease in the abundance of Bacteroidetes, and these changes result in a decreased Firmicutes/Bacteroidetes ratio, which can be an effective indicator for successful GTE intervention, which may generate beneficial health effect to human. Meanwhile, the relative abundances of many detected bacteria genera among three HCM vessels changed through the GTE intervention. The overall effects of GTE on gut microbial beta-diversity were observed by multivariate statistical analyses, and the differences in metabolic profiles from different GTE treatment stages were detected. Moreover, we identified several associations between microbial population and microbial metabolites, which may assist us in establishing new hypotheses for future related studies. In summary, our study suggested that the microbial compositional changes induced by GTE also changed their metabolic functions, and consequentially, may change the host metabolism and impact human health.

Green tea extract prevents obesity in male mice by alleviating gut dysbiosis in association with improved intestinal barrier function that limits endotoxin translocation and adipose inflammation.

Gut-derived endotoxin translocation provokes obesity by inducing TLR4/NFκB inflammation. We hypothesized that catechin-rich green tea extract (GTE) would protect against obesity-associated TLR4/NFκB inflammation by alleviating gut dysbiosis and limiting endotoxin translocation. Male C57BL/6J mice were fed a low-fat (LF) or high-fat (HF) diet containing 0% or 2% GTE for 8 weeks. At Week 7, fluorescein isothiocyanate (FITC)-dextran was administered by oral gavage before assessing its serum concentrations as a gut permeability marker. HF-feeding increased (P<.05) adipose mass and adipose expression of genes involved in TLR4/NFκB-dependent inflammation and macrophage activation. GTE attenuated HF-induced obesity and pro-inflammatory gene expression. GTE in HF mice decreased serum FITC-dextran, and attenuated portal vein and circulating endotoxin concentrations. GTE in HF mice also prevented HF-induced decreases in the expression of intestinal tight junction proteins (TJPs) and hypoxia inducible factor-1α while preventing increases in TLR4/NFκB-dependent inflammatory genes. Gut microbial diversity was increased, and the Firmicutes:Bacteroidetes ratio was decreased, in HF mice fed GTE compared with HF controls. GTE in LF mice did not attenuate adiposity but decreased endotoxin and favorably altered several gut bacterial populations. Serum FITC-dextran was correlated with portal vein endotoxin (P<.001; rP=0.66) and inversely correlated with colonic mRNA levels of TJPs (P<.05; rP=-0.38 to -0.48). Colonic TJPs mRNA were inversely correlated with portal endotoxin (P<.05; rP=-0.33 to -0.39). These data suggest that GTE protects against diet-induced obesity consistent with a mechanism involving the gut-adipose axis that limits endotoxin translocation and consequent adipose TLR4/NFκB inflammation by improving gut barrier function.

Headspace Gas Monitoring of Gut Microbiota Using Targeted and Globally Optimized Targeted Secondary Electrospray Ionization Mass Spectrometry.

This study aimed to develop and incorporate a secondary electrospray ionization (SESI) setup in combination with both targeted tandem mass spectrometry (MS/MS) and a hybrid metabolomics technique, globally optimized targeted mass spectrometry (GOT-MS), to sensitively detect volatile metabolites from the headspace of in vitro gut microbial culture in a human colonic model (HCM). Two SESI-tandem mass spectrometry panels with a comparable number of targeted metabolites/features (77 compounds in the targeted SESI-MS/MS panel and 75 features in the SESI-GOT-MS/MS panel) were established. The analytical performance of the SESI-GOT-MS/MS method, as well as its biological capability, were examined and compared with the targeted SESI-MS/MS method. As a result, the SESI-GOT-MS/MS method detected a similar number of metabolic features with good reproducibility (coefficient of variation <10%) compared to the targeted SESI-MS/MS method. Both methods showed a comparable ability to differentiate the gut microbial culture with or without the addition of green tea extract (GTE) to a HCM. The results from analysis of variance (ANOVA) showed that similar numbers of compounds from targeted SESI-MS/MS and metabolic features from SESI-GOT-MS/MS have significant differences when comparing samples collected from different HCM treatment stages. Partial least-squares discriminant analysis (PLS-DA) indicated that both methods could clearly differentiate the stages of GTE treatment. In summary, we demonstrated that SESI-MS/MS in combination with either targeted or GOT approaches can be a useful tool for monitoring gut microbial metabolism and their response to perturbations.

Metabolomics Study Reveals Enhanced Inhibition and Metabolic Dysregulation in Escherichia coli Induced by Lactobacillus acidophilus-Fermented Black Tea Extract.

This study examined the ability of Lactobacillus acidophilus (LA) to ferment black tea extract (BTE) and the enhancement of Escherichia coli cellular uptake of phenolic compounds when these bacteria were incubated with fermented BTE. The inhibitory effects of BTE to E. coli bacteria with and without fermentation were compared. Several intracellular phenolic compounds as well as metabolic profiles of E. coli with and without treatments were also determined using a high-performance liquid chromatography-tandem mass spectrometry-based approach. Our results showed that of three concentrations from the non-fermented BTE treatment, only the extract from the 25 mg/mL tea leaves solution could inhibit E. coli survival, while LA-fermented BTE extract from 5, 10, and 25 mg/mL tea leaves solutions all inhibited E. coli growth significantly. Intracellular concentrations of (+)-catechin-3-gallate/(-)-epicatechin-3-gallate and (+)-catechin/(-)-epicatechin were significantly higher when E. coli was treated with fermented BTE in comparison to non-fermented BTE. Scanning electron microscopy images indicated that the intracellular phenolic compounds inhibited E. coli growth by increasing endogenous oxidative stress. Metabolic profiles of E. coli were also investigated to understand their metabolic response when treated with BTE, and significant metabolic changes of E. coli were observed. Metabolic profile data were further analyzed using partial least squares discriminant analysis to distinguish the fermented BTE treatment group from the control group and the non-fermented BTE treatment group. The results indicated a large-scale E. coli metabolic dysregulation induced by the fermented BTE. Our findings showed that LA fermentation can be an efficient approach to enhance phenolic inhibition of bacterial cells through increased endogenous oxidative stress and dysregulated metabolic activities.

Specificity protein 1 regulates gene expression related to fatty acid metabolism in goat mammary epithelial cells.

Specificity protein 1 (SP1) is a ubiquitous transcription factor that plays an important role in controlling gene expression. Although important in mediating the function of various hormones, the role of SP1 in regulating milk fat formation remains unknown. To investigate the sequence and expression information, as well as its role in modulating lipid metabolism, we cloned SP1 gene from mammary gland of Xinong Saanen dairy goat. The full-length cDNA of the SP1 gene is 4376 bp including 103 bp of 5'UTR, 2358 bp of ORF (HM_236311) and 1915 bp of 3'UTR, which is predicted to encode a 786 amino acids polypeptide. Phylogenetic tree analysis showed that goat SP1 has the closest relationship with sheep, followed by bovines (bos taurus, odobenus and ceratotherium), pig, primates (pongo, gorilla, macaca and papio) and murine (rattus and mus), while the furthest relationship was with canis and otolemur. Expression was predominant in the lungs, small intestine, muscle, spleen, mammary gland and subcutaneous fat. There were no significant expression level differences between the mammary gland tissues collected at lactation and dry-off period. Overexpression of SP1 in goat mammary epithelial cells (GMECs) led to higher mRNA expression level of peroxisome proliferator-activated receptor-γ (PPARγ) and lower liver X receptor α (LXRα) mRNA level, both of which were crucial in regulating fatty acid metabolism, and correspondingly altered the expression of their downstream genes in GMECs. These results were further enhanced by the silencing of SP1. These findings suggest that SP1 may play an important role in fatty acid metabolism.

Integrated plasma and urine metabolomics coupled with HPLC/QTOF-MS and chemometric analysis on potential biomarkers in liver injury and hepatoprotective effects of Er-Zhi-Wan.

Metabolomics techniques are the comprehensive assessment of endogenous metabolites in a biological system and may provide additional insight into the molecular mechanisms. Er-Zhi-Wan (EZW) is a traditional Chinese medicine formula, which contains Fructus Ligustri Lucidi (FLL) and Herba Ecliptae (HE). EZW is widely used to prevent and treat various liver injuries through the nourishment of the liver. However, the precise molecular mechanism of hepatoprotective effects has not been comprehensively explored. Here, an integrated metabolomics strategy was designed to assess the effects and possible mechanisms of EZW against carbon tetrachloride-induced liver injury, a commonly used model of both acute and chronic liver intoxication. High-performance chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QTOF-MS) combined with chemometric approaches including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used to discover differentiating metabolites in metabolomics data of rat plasma and urine. Results indicate six differentiating metabolites, tryptophan, sphinganine, tetrahydrocorticosterone, pipecolic acid, L-2-amino-3-oxobutanoic acid and phosphoribosyl pyrophosphate, in the positive mode. Functional pathway analysis revealed that the alterations in these metabolites were associated with tryptophan metabolism, sphingolipid metabolism, steroid hormone biosynthesis, lysine degradation, glycine, serine and threonine metabolism, and pentose phosphate pathway. Of note, EZW has a potential pharmacological effect, which might be through regulating multiple perturbed pathways to the normal state. Our findings also showed that the robust integrated metabolomics techniques are promising for identifying more biomarkers and pathways and helping to clarify the function mechanisms of traditional Chinese medicine.

Goat liver X receptor α, molecular cloning, functional characterization and regulating fatty acid synthesis in epithelial cells of goat mammary glands.

The liver X receptor α (LXRα) is a nuclear receptor of the transcription factor and is known to play a crucial role in lipid metabolism processes such as bile acid and fatty acid synthesis in humans and rodents. However, very little information is available on the role of LXRα in the regulation of fatty acid synthesis in the goat mammary gland. In this investigation, a cDNA was isolated from the mammary gland of Xinong Saanen dairy goats and designated as goat LXRα. RT-PCR and RACE gave rise to the full-length cDNA of LXRα, which was comprised of 1654 bp and characterized by an ORF of 1344 bp and 5'- and 3'-UTR regions of 150 and 160 bp, respectively. The deduced amino acid sequence encodes 477 amino acids with a predicted molecular weight (MW) of 50.4kDa and a theoretical isoelectric point (pI) of 6.3. Additionally, homology search and sequence multi-alignment indicated that the putative goat LXRα amino acid sequence is very similar to those of cattle, mice, rats, swine, and humans. Bioinformatic predictions demonstrated that the LXRα protein is located in the nucleus, containing characteristic signatures of a nuclear receptor with DNA-binding domain (DBD) and ligand-binding domain (LBD). Real-time quantitative PCR suggested that LXRα was predominantly expressed in the small intestine, liver, spleen and mammary gland. Treatment of goat mammary gland epithelial cells (GMEC) with different concentrations (i.e., 0.01, 0.1, 1 µM) of T0901317, a synthetic agonist of LXRα, resulted in elevated sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) mRNA levels in response to LXRα activation. The association between different T0901317 concentrations and fatty acid composition in GMEC also was examined using gas chromatography (GC). The results showed that activation of LXRα significantly increased GMEC C18:1 and C18:2 contents, but did not affect levels of saturated fatty acids (SFA). These discoveries are consistent with the notion that LXRα plays a key role in controlling lipogenesis and regulating synthesis of unsaturated fatty acids (UFA) in the mammary gland of goats, which may prove useful in regulation of milk fat production.