Uropathogenic Escherichia coli wield enterobactin-derived catabolites as siderophores.

Uropathogenic Escherichia coli (UPEC) secrete multiple siderophore types to scavenge extracellular iron(III) ions during clinical urinary tract infections, despite the metabolic costs of biosynthesis. Here, we find the siderophore enterobactin (Ent) and its related products to be prominent components of the iron-responsive extracellular metabolome of a model UPEC strain. Using defined Ent biosynthesis and import mutants, we identify lower molecular weight dimeric exometabolites as products of incomplete siderophore catabolism, rather than prematurely released biosynthetic intermediates. In E. coli, iron acquisition from iron(III)-Ent complexes requires intracellular esterases that hydrolyze the siderophore. Although UPEC are equipped to consume the products of completely hydrolyzed Ent, we find that Ent and its derivatives may be incompletely hydrolyzed to yield products with retained siderophore activity. These results are consistent with catabolic inefficiency as means to obtain more than one iron ion per siderophore molecule. This is compatible with an evolved UPEC strategy to maximize the nutritional returns from metabolic investments in siderophore biosynthesis.

Exploring the Exo-Metabolomes and Volatile and Non-Volatile Compounds of Metarhizium carneum and Lecanicillium uredinophilum.

Efforts are intensifying to identify bioactive microbial metabolites from biocontrol agents to manage plant pathogens in critical crops. This study examined both volatile organic compounds (VOCs) and non-volatile compounds from Metarhizium carneum and Lecanicillium uredinophilum strains for their antimicrobial effects against various phytopathogens and analyzed their exo-metabolomes. M. carneum VOCs inhibited four bacterial and eight fungal species by up to 45.45%, while L. uredinophilum VOCs inhibited five bacterial and eight fungal species by up to 50.91%. Additionally, n-BuOH extracts from both biocontrol agents effectively targeted three fungi and five bacteria. The exo-metabolomes of M. carneum and L. uredinophilum included 125 and 102 spectrometric features, respectively, primarily consisting of polyketides, alkaloids, lipids, organic aromatic compounds, terpenoids, and peptides. Our findings revealed a correlation between the phylogenetic relationships of M. carneum strains, their bioactivity patterns against phytopathogens, and their metabolomic profiles. Notably, some compounds detected in both fungi previously demonstrated biological activity against plant pathogens, enhancing their biocontrol potential. This study not only evidences the antimicrobial properties of diffusible compounds from M. carneum and L. uredinophilum, but also documents the antimicrobial potential of their VOCs for the first time, supporting their use in sustainable agricultural practices, reducing reliance on chemical inputs.

Uncovering Metabolic Alterations in HCT-116 Colon Cancer Cells upon Exposure to Bamboo Leaf Extract Obtained from Guadua incana Londoño.

Metabolic alterations are increasingly recognized as important aspects of colorectal cancer (CRC), offering potential avenues for identifying therapeutic targets. Previous studies have demonstrated the cytotoxic potential of bamboo leaf extract obtained from Guadua incana (BLEGI) against HCT-116 colon cancer cells. However, the altered metabolic pathways in these tumor cells remain unknown. Therefore, this study aimed to employ an untargeted metabolomic approach to reveal the metabolic alterations of the endometabolome and exometabolome of HCT-116 cells upon exposure to BLEGI treatment. First, a chemical characterization of the BLEGI was conducted through liquid chromatography coupled with mass spectrometry (LC-MS). Next, we assessed cell viability via MTT and morphological analysis using an immunofluorescence assay against colon cancer cells, and anti-inflammatory activity using an LPS-stimulated macrophage model. Subsequently, we employed LC-MS and proton nuclear magnetic resonance (1H-NMR) to investigate intra- and extracellular changes. Chemical characterization primarily revealed the presence of compounds with a flavone glycoside scaffold. Immunofluorescence analysis showed condensed chromatin and subsequent formation of apoptotic bodies, suggesting cell death by apoptosis. The results of the metabolomic analysis showed 98 differential metabolites, involved in glutathione, tricarboxylic acid cycle, and lipoic acid metabolism, among others. Additionally, BLEGI demonstrated significant nitric oxide (NO) inhibitory capacity in macrophage cells. This study enhances our understanding of BLEGI's possible mechanism of action and provides fresh insights into therapeutic targets for treating this disease.

Balancing Trade-Offs Imposed by Growth Media and Mass Spectrometry for Bacterial Exometabolomics.

The bacterial exometabolome consists of a vast array of specialized metabolites, many of which are only produced in response to specific environmental stimuli. For this reason, it is desirable to control the extracellular environment with a defined growth medium composed of pure ingredients. However, complex (undefined) media are expected to support the robust growth of a greater variety of microorganisms than defined media. Here, we investigate the trade-offs inherent to a range of complex and defined solid media for the growth of soil microorganisms, production of specialized metabolites, and detection of these compounds using direct infusion mass spectrometry. We find that complex media support growth of more soil microorganisms, as well as allowing for the detection of more previously discovered natural products as a fraction of total m/z features detected in each sample. However, the use of complex media often caused mass spectrometer injection failures and poor-quality mass spectra, which in some cases resulted in over a quarter of samples being removed from analysis. Defined media, while more limiting in growth, generated higher quality spectra and yielded more m/z features after background subtraction. These results inform future exometabolomic experiments requiring a medium that supports the robust growth of many soil microorganisms. IMPORTANCE Bacteria are capable of producing and secreting a rich diversity of specialized metabolites. Yet, much of their exometabolome remains hidden due to challenges associated with eliciting specialized metabolite production, labor-intensive sample preparation, and time-consuming analysis techniques. Using our versatile three-dimensional (3D)-printed culturing platform, SubTap, we demonstrate that rapid exometabolomic data collection from a diverse set of environmental bacteria is feasible. We optimized our platform by surveying Streptomyces isolated from soil on a variety of media types to assess viability, degree of specialized metabolite production, and compatibility with downstream LESA-DIMS analysis. Ultimately, this will enable data-rich experimentation, allowing for a better understanding of bacterial exometabolomes.

Sunitinib versus Pazopanib Dilemma in Renal Cell Carcinoma: New Insights into the In Vitro Metabolic Impact, Efficacy, and Safety.

Sunitinib and pazopanib are tyrosine kinase inhibitors (TKIs) used as first-line therapy for metastatic renal cell carcinoma (RCC). Although these TKIs are associated with similar survival outcomes, some differences have been reported in their safety profiles. In this work, traditional toxicological endpoints (cell viability and growth, oxidative stress, and nuclear morphology) and 1H NMR spectroscopy-based metabolomics analysis were used to provide new insights into the cytotoxicity and metabolic mechanisms underlying sunitinib and pazopanib treatments. Tumoral (Caki-1) and non-tumoral (HK-2) human renal cells were exposed to clinically relevant concentrations of sunitinib (2 µM) or pazopanib (50 µM). Sunitinib showed selectivity for cancer cells, inhibiting proliferation, and inducing apoptotic death of Caki-1 cells, whereas pazopanib had a similar cytotoxic effect in both tumoral and non-tumoral cells. 1H-NMR metabolomics unveiled a higher impact of sunitinib on the levels of intracellular metabolites of Caki-1 cells (seven dysregulated metabolites), suggesting dysregulations on amino acid, glutathione and glycerophospholipid metabolisms. In contrast, pazopanib had a higher impact on the levels of extracellular metabolites of Caki-1 cells (seven dysregulated metabolites in culture medium), unveiling alterations on amino acid and energetic metabolisms. In HK-2 cells, sunitinib caused only a minor increase in intracellular isoleucine levels, whereas pazopanib induced several alterations on the intracellular (three dysregulated metabolites) and extracellular (three dysregulated metabolites) compartments suggesting changes on amino acid, glycerophospholipid, and energy metabolisms. Our results demonstrate that these TKIs elicit distinct cellular and metabolic responses, with sunitinib showing better in vitro efficacy against target RCC cells and lesser nephrotoxic potential than pazopanib.

Hypersensitive SSY1 mutations negatively influence transition to quiescence in yeast Saccharomyces cerevisiae.

Most cells spend the majority of their life in the non-proliferating, quiescent state. Transition to this state is crucial for microorganisms to survive long starvation periods and restart divisions afterwards. Experimental evolution allowed us to identify several mutation in genes that are presumably important for such transition in yeast cells. Most of these candidate genes belong to the SPS amino acid sensing pathway or to the SIR complex. We assembled these mutations on the ancestral strain background. Analysis of the quiescent/non-quiescent cell ratio of the starved yeast populations confirmed the crucial role of SSY1, the primary receptor component of the SPS sensor, in transition to the Q state. The evolved SSY1 mutations increased yeast sensitivity to amino acid presence in the environment. This resulted in decreased quiescent cell fraction and a 5.14% increase of the total amino acid content in the starved populations. We discuss external amino acid sensing via the SPS pathway as one of the mechanisms influencing transition to quiescence.

Periphytic Microbial Response to Environmental Phosphate (P) Bioavailability and Its Relevance to P Management in Paddy Fields.

Periphyton occurs widely in shallow-water ecosystems such as paddy fields and plays a critical part in regulating local phosphorus cycling. As such, understanding the mechanisms of biofilms' response to environmental phosphate (P) variability may lead to better perceptions of P utilization and retention in rice farms. The present study aims at exploring the biological and biochemical processes underlying periphyton's P buffering capability through examining changes in community structure, phosphorus uptake and storage, and molecular makeup of the exometabolome at different levels of P availability. Under stressed (both excessive and scarce) phosphorus conditions, we found increased populations of bacterial genera capable of transforming orthophosphate to polyphosphate, as well as mixotrophic algae, that can survive through phagotrophy. These results were corroborated by observed polyphosphate buildup under low- and high-P treatment. Exometabolomic analyses further revealed that periphytic organisms may substitute sulfur (S)-containing lipids for phospholipids, use siderophores to dissolve iron (hydr)oxides to scavenge adsorbed P, and synthesize auxins to resist phosphorus starvation. These findings not only shed light on the mechanistic insights responsible for driving the periphytic P buffer but attest to the ecological roles of periphyton in aiding plants such as rice to overcome P limitations in the natural environment. IMPORTANCE The ability of periphyton to buffer environmental P in shallow aquatic ecosystems may be a natural lesson on P utilization and retention in paddy fields. This work revealed the routes and tools through which periphytic organisms adapt to and regulate ambient P fluctuation. The mechanistic understanding further implicates that the biofilm may serve rice plants to alleviate P stress. Additional results from extracellular metabolite analyses suggest the dissolved periphytic exometabolome can be a valuable nutrient source for soil microbes and plants to reduce biosynthetic costs. These discoveries have the potential to improve our understanding of biogeochemical cycling of phosphorus in general and to refine P management strategies for rice farms in particular.

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures.

Biological organisms are constantly exposed to an immense repertoire of molecules that cover environmental or food-derived molecules and drugs, triggering a continuous flow of stimuli-dependent adaptations. The diversity of these chemicals as well as their concentrations contribute to the multiplicity of induced effects, including activation, stimulation, or inhibition of physiological processes and toxicity. Metabolism, as the foremost phenotype and manifestation of life, has proven to be immensely sensitive and highly adaptive to chemical stimuli. Therefore, studying the effect of endo- or xenobiotics over cellular metabolism delivers valuable knowledge to apprehend potential cellular activity of individual molecules and evaluate their acute or chronic benefits and toxicity. The development of modern metabolomics technologies such as mass spectrometry or nuclear magnetic resonance spectroscopy now offers unprecedented solutions for the rapid and efficient determination of metabolic profiles of cells and more complex biological systems. Combined with the availability of well-established cell culture techniques, these analytical methods appear perfectly suited to determine the biological activity and estimate the positive and negative effects of chemicals in a variety of cell types and models, even at hardly detectable concentrations. Metabolic phenotypes can be estimated from studying intracellular metabolites at homeostasis in vivo, while in vitro cell cultures provide additional access to metabolites exchanged with growth media. This article discusses analytical solutions available for metabolic phenotyping of cell culture metabolism as well as the general metabolomics workflow suitable for testing the biological activity of molecular compounds. We emphasize how metabolic profiling of cell supernatants and intracellular extracts can deliver valuable and complementary insights for evaluating the effects of xenobiotics on cellular metabolism. We note that the concepts and methods discussed primarily for xenobiotics exposure are widely applicable to drug testing in general, including endobiotics that cover active metabolites, nutrients, peptides and proteins, cytokines, hormones, vitamins, etc.

Algae-bacteria interactions that balance the planktonic microbiome.

Phytoplankton communities within the photic zones of the oceans and lakes are characterised by highly complex assemblages of unicellular microalgae and associated bacteria. The interconnected evolutionary history of algae and bacteria allowed the formation of a wide spectrum of associations defined by orchestrated nutrient exchange, mutual support with growth factors, quorum sensing mediation, and episodic killing of the partners to obtain more resources. In this review, we discuss how these cross-kingdom interactions shape plankton communities that undergo annual, seasonal switching between alternative states with balanced multispecies consortia. We illustrate how these microscopic interactions can have consequences that scale up to influence global element cycling.

Fungal biotransformation of short-chain n-alkylcycloalkanes.

The cycloalkanes, comprising up to 45% of the hydrocarbon fraction, occur in crude oil or refined oil products (e.g., gasoline) mainly as alkylated cyclohexane derivatives and have been increasingly found in environmental samples of soil and water. Furthermore, short-chain alkylated cycloalkanes are components of the so-called volatile organic compounds (VOCs). This study highlights the biotransformation of methyl- and ethylcyclohexane by the alkane-assimilating yeast Candida maltosa and the phenol- and benzoate-utilizing yeast Trichosporon mucoides under laboratory conditions. In the course of this biotransformation, we detected 25 different metabolites, which were analyzed by HPLC and GC-MS. The biotransformation process of methylcyclohexane in both yeasts involve (A) ring hydroxylation at different positions (C2, C3, and C4) and subsequent oxidation to ketones as well as (B) oxidation of the alkyl side chain to hydroxylated and acid products. The yeast T. mucoides additionally performs ring hydroxylation at the C1-position and (C) oxidative decarboxylation and (D) aromatization of cyclohexanecarboxylic acid. Both yeasts also oxidized the saturated ring system and the side chain of ethylcyclohexane. However, the cyclohexylacetic acid, which was formed, seemed not to be substrate for aromatization. This is the first report of several new transformation reactions of alkylated cycloalkanes for eukaryotic microorganisms.

New insight on obesity and adipose-derived stem cells using comprehensive metabolomics.

Obesity affects the functional capability of adipose-derived stem cells (ASCs) and their effective use in regenerative medicine through mechanisms that are still poorly understood. In the present study we used a multiplatform [LC/MS, GC/MS and capillary electrophoresis/MS (CE/MS)], metabolomics, untargeted approach to investigate the metabolic alteration underlying the inequalities observed in obesity-derived ASCs. The metabolic fingerprint (metabolites within the cells) and footprint (metabolites secreted in the culture medium), from obesity- and non-obesity-derived ASCs of humans or mice, were characterized to provide valuable information. Metabolites associated with glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and the polyol pathway were increased in the footprint of obesity-derived human ASCs, indicating alterations in carbohydrate metabolism, whereas, from the murine model, deep differences in lipid and amino acid catabolism were highlighted. Therefore, new insights on the ASCs' metabolome were provided that enhance our understanding of the processes underlying ASCs' stemness capacity and its relationship with obesity, in different cell models.

Proteins involved in wine aroma compounds metabolism by a Saccharomyces cerevisiae flor-velum yeast strain grown in two conditions.

A proteomic and exometabolomic study was conducted on Saccharomyces cerevisiae flor yeast strain growing under biofilm formation condition (BFC) with ethanol and glycerol as carbon sources and results were compared with those obtained under no biofilm formation condition (NBFC) containing glucose as carbon source. By using modern techniques, OFFGEL fractionator and LTQ-Orbitrap for proteome and SBSE-TD-GC-MS for metabolite analysis, we quantified 84 proteins including 33 directly involved in the metabolism of glycerol, ethanol and 17 aroma compounds. Contents in acetaldehyde, acetic acid, decanoic acid, 1,1-diethoxyethane, benzaldehyde and 2-phenethyl acetate, changed above their odor thresholds under BFC, and those of decanoic acid, ethyl octanoate, ethyl decanoate and isoamyl acetate under NBFC. Of the twenty proteins involved in the metabolism of ethanol, acetaldehyde, acetoin, 2,3-butanediol, 1,1-diethoxyethane, benzaldehyde, organic acids and ethyl esters, only Adh2p, Ald4p, Cys4p, Fas3p, Met2p and Plb1p were detected under BFC and as many Acs2p, Ald3p, Cem1p, Ilv2p, Ilv6p and Pox1p, only under NBFC. Of the eight proteins involved in glycerol metabolism, Gut2p was detected only under BFC while Pgs1p and Rhr2p were under NBFC. Finally, of the five proteins involved in the metabolism of higher alcohols, Thi3p was present under BFC, and Aro8p and Bat2p were under NBFC.

Deorphanizing solute carriers in Saccharomyces cerevisiae for secondary uptake of xenobiotic compounds.

The exchange of small molecules between the cell and the environment happens through transporter proteins. Besides nutrients and native metabolic products, xenobiotic molecules are also transported, however it is not well understood which transporters are involved. In this study, by combining exo-metabolome screening in yeast with transporter characterization in Xenopus oocytes, we mapped the activity of 30 yeast transporters toward six small non-toxic substrates. Firstly, using LC-MS, we determined 385 compounds from a chemical library that were imported and exported by S. cerevisiae. Of the 385 compounds transported by yeast, we selected six compounds (viz. sn-glycero-3-phosphocholine, 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, acrylic acid, 2-benzoxazolol) for characterization against 30 S. cerevisiae xenobiotic transport proteins expressed in Xenopus oocytes. The compounds were selected to represent a diverse set of chemicals with a broad interest in applied microbiology. Twenty transporters showed activity toward one or more of the compounds. The tested transporter proteins were mostly promiscuous in equilibrative transport (i.e., facilitated diffusion). The compounds 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, and sn-glycero-3-phosphocholine were transported equilibratively by transporters that could transport up to three of the compounds. In contrast, the compounds acrylic acid and 2-benzoxazolol, were strictly transported by dedicated transporters. The prevalence of promiscuous equilibrative transporters of non-native substrates has significant implications for strain development in biotechnology and offers an explanation as to why transporter engineering has been a challenge in metabolic engineering. The method described here can be generally applied to study the transport of other small non-toxic molecules. The yeast transporter library is available at AddGene (ID 79999).

Indole modulates cooperative protein-protein interactions in the flagellar motor.

Indole is a major component of the bacterial exometabolome, and the mechanisms for its wide-ranging effects on bacterial physiology are biomedically significant, although they remain poorly understood. Here, we determined how indole modulates the functions of a widely conserved motility apparatus, the bacterial flagellum. Our experiments in Escherichia coli revealed that indole influences the rotation rates and reversals in the flagellum's direction of rotation via multiple mechanisms. At concentrations higher than 1 mM, indole decreased the membrane potential to dissipate the power available for the rotation of the motor that operates the flagellum. Below 1 mM, indole did not dissipate the membrane potential. Instead, experiments and modeling indicated that indole weakens cooperative protein interactions within the flagellar complexes to inhibit motility. The metabolite also induced reversals in the rotational direction of the motor to promote a weak chemotactic response, even when the chemotaxis response regulator, CheY, was lacking. Experiments further revealed that indole does not require the transporter Mtr to cross the membrane and influence motor functions. Based on these findings, we propose that indole modulates intra- and inter-protein interactions in the cell to influence several physiological functions.

Investigation of the Exometabolomic Profiles of Rat Islets of Langerhans Cultured in Microfluidic Biochip.

Diabetes mellitus (DM) is a complex disease with high prevalence of comorbidity and mortality. DM is predicted to reach more than 700 million people by 2045. In recent years, several advanced in vitro models and analytical tools were developed to investigate the pancreatic tissue response to pathological situations and identify therapeutic solutions. Of all the in vitro promising models, cell culture in microfluidic biochip allows the reproduction of in-vivo-like micro-environments. Here, we cultured rat islets of Langerhans using dynamic cultures in microfluidic biochips. The dynamic cultures were compared to static islets cultures in Petri. The islets' exometabolomic signatures, with and without GLP1 and isradipine treatments, were characterized by GC-MS. Compared to Petri, biochip culture contributes to maintaining high secretions of insulin, C-peptide and glucagon. The exometabolomic profiling revealed 22 and 18 metabolites differentially expressed between Petri and biochip on Day 3 and 5. These metabolites illustrated the increase in lipid metabolism, the perturbation of the pentose phosphate pathway and the TCA cycle in biochip. After drug stimulations, the exometabolome of biochip culture appeared more perturbed than the Petri exometabolome. The GLP1 contributed to the increase in the levels of glycolysis, pentose phosphate and glutathione pathways intermediates, whereas isradipine led to reduced levels of lipids and carbohydrates.

Untargeted metabolite profiling to elucidate rhizosphere and leaf metabolome changes of wheat cultivars (Triticum aestivum L.) treated with the plant growth-promoting rhizobacteria Paenibacillus alvei (T22) and Bacillus subtilis.

The rhizosphere is a highly complex and biochemically diverse environment that facilitates plant-microbe and microbe-microbe interactions, and this region is found between plant roots and the bulk soil. Several studies have reported plant root exudation and metabolite secretion by rhizosphere-inhabiting microbes, suggesting that these metabolites play a vital role in plant-microbe interactions. However, the biochemical constellation of the rhizosphere soil is yet to be fully elucidated and thus remains extremely elusive. In this regard, the effects of plant growth-promoting rhizobacteria (PGPR)-plant interactions on the rhizosphere chemistry and above ground tissues are not fully understood. The current study applies an untargeted metabolomics approach to profile the rhizosphere exo-metabolome of wheat cultivars generated from seed inoculated (bio-primed) with Paenibacillus (T22) and Bacillus subtilis strains and to elucidate the effects of PGPR treatment on the metabolism of above-ground tissues. Chemometrics and molecular networking tools were used to process, mine and interpret the acquired mass spectrometry (MS) data. Global metabolome profiling of the rhizosphere soil of PGPR-bio-primed plants revealed differential accumulation of compounds from several classes of metabolites including phenylpropanoids, organic acids, lipids, organoheterocyclic compounds, and benzenoids. Of these, some have been reported to function in plant-microbe interactions, chemotaxis, biocontrol, and plant growth promotion. Metabolic perturbations associated with the primary and secondary metabolism were observed from the profiled leaf tissue of PGPR-bio-primed plants, suggesting a distal metabolic reprograming induced by PGPR seed bio-priming. These observations gave insights into the hypothetical framework which suggests that PGPR seed bio-priming can induce metabolic changes in plants leading to induced systemic response for adaptation to biotic and abiotic stress. Thus, this study contributes knowledge to ongoing efforts to decipher the rhizosphere metabolome and mechanistic nature of biochemical plant-microbe interactions, which could lead to metabolome engineering strategies for improved plant growth, priming for defense and sustainable agriculture.

Analysis of the Metabolic Response of Planktonic Cells and Biofilms of Klebsiella pneumoniae to Sublethal Disinfection with Sodium Hypochlorite Measured by NMR.

Klebsiella pneumoniae is a pathogenic agent able to form biofilms on water storage tanks and pipe walls. This opportunistic pathogen can generate a thick layer as one of its essential virulence factors, enabling the bacteria to survive disinfection processes and thus develop drug resistance. Understanding the metabolic differences between biofilm and planktonic cells of the K. pneumoniae response to NaClO is key to developing strategies to control its spread. In this study, we performed an NMR metabolic profile analysis to compare the response to a sublethal concentration of sodium hypochlorite of biofilm and planktonic cells of K. pneumoniae cultured inside silicone tubing. Metabolic profiles revealed changes in the metabolism of planktonic cells after a contact time of 10 min with 7 mg L-1 of sodium hypochlorite. A decrease in the production of metabolites such as lactate, acetate, ethanol, and succinate in this cell type was observed, thus indicating a disruption of glucose intake. In contrast, the biofilms displayed a high metabolic heterogeneity, and the treatment did not affect their metabolic signature.

Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids.

The interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex in vitro systems, culminating with the use of the physiological relevant human colonoids as a model to study the interactions between EHEC and different gut commensals. We demonstrated that Bacteroides thetaiotaomicron and Enterococcus faecalis enhance virulence expression and AE lesion formation in cultured epithelial cells, as well as on the colonic epithelium, while commensal E. coli did not affect these phenotypes. Importantly, in the presence of these three commensals together, virulence and AE lesion are enhanced. Moreover, we identified specific changes in the metabolic landscape promoted by different members of the gut microbiota and showed that soluble factors released by E. faecalis can increase EHEC virulence gene expression. Our study highlights the importance of interspecies bacterial interactions and chemical exchange in the modulation of EHEC virulence. IMPORTANCE Enterohemorrhagic E. coli (EHEC) is a natural human pathogen that poorly colonizes mice. Hence, the use of murine models to understand features of EHEC infection is a challenge. In this study, we use human colonoids as a physiologically relevant model to study interactions between EHEC and gut commensals. We demonstrate that the ability of EHEC to form AE lesions on the intestinal epithelium is enhanced by the presence of certain gut commensals, such as B. thetaiotaomicron and E. faecalis, while it is not affected by commensal E. coli. Furthermore, we show that commensal bacteria differently impact the metabolic landscape. These data suggest that microbiota compositions can differentially modulate EHEC-mediated disease.

Fast Sampling of the Cellular Metabolome.

Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally, a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample. An issue which has often been overlooked in microbial metabolomics is the fact that many intracellular metabolites are also present in significant amounts outside the cells and may interfere with the quantification of the endo metabolome. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, this release can be minimized by adaptation of the quenching method. For prokaryotic cells, this has not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach. Here we present an overview of different sampling, quenching, and extraction methods developed for microbial metabolomics, described in the literature. Detailed protocols are provided for rapid sampling, quenching, and extraction, for measurement of metabolites in total broth samples, washed cell samples, and supernatant, to be applied for quantitative metabolomics of both eukaryotic and prokaryotic microorganisms.

Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation.

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.