Presence Of Trimethylamine N-oxide Research Articles

Small organic osmolytes accelerate actin filament assembly and stiffen filaments.

Actin filament assembly and mechanics are crucial for maintenance of cell structure, motility, and division. Actin filament assembly occurs in a crowded intracellular environment consisting of various types of molecules, including small organic molecules known as osmolytes. Ample evidence highlights the protective functions of osmolytes such as trimethylamine-N-oxide (TMAO), including their effects on protein stability and their ability to counteract cellular osmotic stress. Yet, how TMAO affects individual actin filament assembly dynamics and mechanics is not well understood. We hypothesize that, owing to its protective nature, TMAO will enhance filament dynamics and stiffen actin filaments due to increased stability. In this study, we investigate osmolyte-dependent actin filament assembly and bending mechanics by measuring filament elongation rates, steady-state filament lengths, and bending persistence lengths in the presence of TMAO using total internal reflection fluorescence microscopy and pyrene assays. Our results demonstrate that TMAO increases filament elongation rates as well as steady-state average filament lengths, and enhances filament bending stiffness. Together, these results will help us understand how small organic osmolytes modulate cytoskeletal protein assembly and mechanics in living cells.

Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Effect of Ligand Binding on Polymer Diffusiophoresis

Diffusiophoresis is the migration of a macromolecule in response to a concentration gradient of a cosolute in liquids. Diffusiophoresis of polyethylene glycol (PEG) in water occurs from high to low concentration of trimethylamine-N-oxide (TMAO). This is consistent with the preferential hydration of PEG observed in the presence of TMAO. In other words, PEG migrates in the direction in which it lowers its chemical potential. On the other hand, although PEG is found to preferentially bind urea in water, PEG diffusiophoresis still occurs from high to low urea concentration. Thus, PEG migrates in the direction that increases its chemical potential in the urea case. Here, a ligand-binding model for polymer diffusiophoresis in the presence of a cosolute that preferentially binds to polymer is developed. It includes both actual polymer–ligand binding and the effect of the polymer on cosolute thermodynamic activity. This model shows that polymer–cosolute binding has a marginal effect on polymer diffusiophoresis and indicates that weak repulsive interactions, such as hard-core exclusion forces, are the main factor responsible for the observed PEG diffusiophoresis from high to low urea concentration. This work contributes to a better understanding of diffusiophoresis of macromolecules in response to gradients of nonelectrolytes.

Counteracting Effects of Trimethylamine N-Oxide against Urea in Aqueous Solutions: Insights from Theoretical Two-Dimensional Infrared Spectroscopy.

The study of small osmolytes in their aqueous solutions has gained significant attention because of their relevance to structure and thermodynamics of proteins in aqueous media. Special attention has been given to the binary and ternary aqueous solutions of urea and trimethylamine N-oxide (TMAO). Urea is a well-known protein denaturant, while TMAO protects proteins in their native states. Interestingly, TMAO counteracts urea's ability to denature proteins when present in solutions with approximately half of the concentration of urea. Vibrational spectroscopy can improve our understanding of the molecular origin of this counteracting effect because of its sensitivity to local structure and dynamics. We present results of theoretical linear vibrational and two-dimensional infrared (2DIR) spectroscopy of water in the binary and ternary aqueous solutions of TMAO and urea. The 2DIR spectra are calculated using the electronic structure/molecular dynamics approach. The non-Condon effects in spectral transitions are incorporated in the theoretical calculations of 2DIR spectra. It is found that TMAO disrupts the local structure of water, while urea leaves it essentially unaffected. The 2DIR results show that both TMAO and urea slow down the dynamics of spectral diffusion of water. The extent of slowing down is found to be particularly significant for both hydration and bulk water in the presence of TMAO which can be attributed to strong hydrogen bonds between the water and TMAO molecules. The water molecules present in the hydration layer of the solutes in the ternary solutions are found to relax at even slower rates compared to that in their binary solutions in water. The hydrogen bonds between TMAO and urea are found to be not stable. Thus, the counteracting effect of TMAO against urea is seen to take place mainly through water-mediated interactions. Such TMAO-induced effects giving rise to more structured and slower hydrogen-bonded network are successfully captured through 2DIR spectroscopic calculations.

TMAO: Protecting proteins from feeling the heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 μs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules “herding” water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

TMAO Destabilizes RNA Secondary Structure via Direct Hydrogen Bond Interactions.

Trimethylamine N-oxide (TMAO) is an osmolyte that accumulates in cells in response to osmotic stress. TMAO stabilizes proteins by the entropic stabilization mechanism, which pictures TMAO as a nanocrowder that predominantly destabilizes the unfolded state. However, the mechanism of action of TMAO on RNA is much less understood. Here, we use all-atom molecular dynamics simulations to investigate how TMAO interacts with a 12-nt RNA hairpin with a high melting temperature, and an 8-nt RNA hairpin, which has a relatively fluid native basin in the absence of TMAO. The use of the two hairpins with different free energy of stabilization allows us to probe the origin of the destabilization effect of TMAO on RNA molecules without the possibility of forming tertiary interactions. We generated multiple trajectories using all-atom molecular dynamics (MD) simulations in explicit water by employing AMBER and CHARMM force fields, both in the absence and presence of TMAO. We observed qualitatively similar RNA-TMAO interaction profiles from the simulations using the two force fields. TMAO hydrogen bond interactions are largely depleted around the paired RNA bases and ribose sugars. In contrast, we show that the oxygen atom in TMAO, the hydrogen bond acceptor, preferentially interacts with the hydrogen bond donors in the solvent exposed bases, such as those in the stem-loop and the destabilized base stacks in the unfolded state, especially in the marginally stable 8-nt RNA hairpin. The predicted destabilization mechanism through TMAO-RNA hydrogen bond interactions could be tested using two-dimensional IR spectroscopy. Since TMAO does not significantly interact with the hydroxyl group of the ribose sugars, we predict that similar results must also hold for DNA.

Basket-type G-quadruplex with two tetrads in the presence of TMAO and urea: A molecular dynamics study

DNA G-quadruplexes are non-canonical DNA structures, which have been proved to play an important role in key biological processes, such as gene expression, replication, regulation or telomere maintenance. Due to their presence in promoter regions of many oncogenes, G-quadruplexes are also considered as potential target for cancer therapies. Besides their biological implications, G-quadruplexes are present in genomes of various organisms, including deep sea piezophilic microorganisms, who adopt certain levels of co-solutes to protect their internal structures against high pressures. In this work, we study a stable DNA G-quadruplex with only two tetrads in presence of co-solutes. By means of molecular dynamics (MD) simulations, we elucidate the alterations in structural features and solvation properties in pure water and in the presence of physiological levels of trimethylamine-N-oxide (TMAO), urea and combined TMAO:urea solutions. Our simulations unravel the nature of nucleic acid interactions with both co-solutes and water in terms of their impact on the flexibility of the local structure, formation of inter- and intramolecular hydrogen bonds, non-bonded contacts and the location of co-solute molecules around the G-quadruplex.

Influence of TMAO and Pressure on the Folding Equilibrium of TrpCage.

Trimethylamine-N-oxide (TMAO) is an osmolyte known for its ability to counteract the pressure denaturation of proteins. Computational studies addressing the molecular mechanisms of TMAO's osmolyte action have however focused exclusively on its protein-stabilizing properties at ambient pressure, neglecting the changes that may occur under high-pressure conditions where TMAO's hydration structure changes to that of increased water binding. Here, we present the first study on the combined effect of pressure and TMAO on a mini-protein, TrpCage. The results showed that at high pressures, nonpolar residues packed less tightly and the salt bridge of TrpCage was destabilized. This effect was mitigated by TMAO which was found to be strongly depleted from the protein/water interface at 1 kbar than at 1 bar ambient pressure, thus counterbalancing the thermodynamically unfavorable effect of elevated pressure in the free energy of folding. TMAO was depleted from charged groups, like the salt bridge-forming ones, and accumulated around hydrophobic groups. Still, it stabilized both kinds of interactions. Furthermore, enthalpically favorable TrpCage-water hydrogen bonds were reduced in the presence of TMAO, causing a stronger destabilization of the unfolded state than the folded state. This shifted the protein-folding equilibrium toward the folded state. Therefore, TMAO showed stabilizing effects on different kinds of groups, which were partially enhanced at high pressures.

Controlling the Rigidity of Kinesin-Propelled Microtubules in an In Vitro Gliding Assay Using the Deep-Sea Osmolyte Trimethylamine N-Oxide.

The biomolecular motor protein kinesin and its associated filamentous protein microtubule have been finding important nanotechnological applications in the recent years. Rigidity of the microtubules, which are propelled by kinesin motors in an in vitro gliding assay, is an important metric that determines the success of utilization of microtubules and kinesins in various applications, such as transportation, sensing, sorting, molecular robotics, etc. Therefore, regulating the rigidity of kinesin-propelled microtubules has been critical. In this work, we report a simple strategy to regulate the rigidity of kinesin-propelled microtubules in an in vitro gliding assay. We demonstrate that rigidity of the microtubules, propelled by kinesins in an in vitro gliding assay, can be modulated simply by using the natural osmolyte trimethylamine N-oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of microtubules can be modulated over a wide range. Based on this strategy, we are able to reduce the persistence length of microtubules, a measure of microtubule rigidity, ∼8 fold by using TMAO at the concentration of 1.5 M. Furthermore, we found that the decreased rigidity of the kinesin-propelled microtubules can be restored upon elimination of TMAO from the in vitro gliding assay. Alteration in the rigidity of microtubules is accounted for by the non-uniformity of the force applied by kinesins along the microtubules in the presence of TMAO. This work offers a facile strategy to reversibly regulate the rigidity of kinesin-propelled microtubules in situ, which would widen the applications of the biomolecular motor kinesin and its associated protein microtubule in various fields.

PREPARATION, CHARACTERIZATION AND ANTIBACTERIAL STUDIES OF HYDRAZONE SUBSTITUTED MOLYBDENUM CARBONYLS

The hydrazones; (E)-N'-(3-methoxybenzylidene)nicotinohydrazide (ANH) and (E)-N'-(2-hydroxybenzylidene)isonicotinohydrazide (SIH) were prepared by condensation reaction between either m-anisaldehyde or salicylaldehyde with nicotinic hydrazide and isoniazid respectively. Substituting CO in Mo hexacarbonyl with the hydrazones in the presence of trimethylamine N-oxide as decarbonylating agent at ambient temperature under inert atmosphere produced tricarbonyl constituents. The complexes were characterized via spectroscopic techniques, elemental analysis and evaluated for antibacterial mode and action. FTIR/Raman, 1H and 13C MMR spectra obtained revealed that three CO were substituted for hydrazones in the hexacarbonyl, consistent with CHN elemental analysis. The ancillary ligands coordinate through the pyrimidine nitrogen to the Mo centre in a meridional geometry; Mo(CO)3(ANH)3 and Mo(CO)3(SIH)3. The coordination mode afforded a fairly stable complex which released CO in DMSO with release rate of 1.9 × 10-3 and 1.07 × 10-2 kCO) s-1. Examination of compounds ability to release CO to myoglobin under physiological pH revealed that carbonmonoxyhemoglobin was spontaneously formed at 3 to 4 seconds of introduction of the complexes. Antibacterial action against both Gram-negative and -positive bacteria revealed that Mo(CO)3(ANH)3 shows both bacteriostatic and bactericidal action against E coli, S. aureus and P. aeruginosa with activity comparable to the ampicillin control. The Mo(CO)3(SIH)3 analogue was however not effective in inhibiting the growth of the organisms at concentrations below 1000 µM . The methoxy and hydroxyl group on the ligand is suspected to account for the differences in action. The hydrophilic hydroxyl group in Mo(CO)3(SIH)3 can hinder it cell wall penetrating ability compared to the lipophilic group present in Mo(CO)3(ANH)3.

Gut microbiota derived trimethylamine N-oxide (TMAO) detection through molecularly imprinted polymer based sensor

Trimethylamine N-oxide (TMAO), a microbiota-derived metabolite has been implicated in human health and disease. Its early detection in body fluids has been presumed to be significant in understanding the pathogenesis and treatment of many diseases. Hence, the development of reliable and rapid technologies for TMAO detection may augment our understanding of pathogenesis and diagnosis of diseases that TMAO has implicated. The present work is the first report on the development of a molecularly imprinted polymer (MIP) based electrochemical sensor for sensitive and selective detection of TMAO in body fluids. The MIP developed was based on the polypyrrole (PPy), which was synthesized via chemical oxidation polymerization method, with and without the presence of TMAO. The MIP, NIP and the non-sonicated polymer (PPy-TMAO) were separately deposited electrophoretically onto the hydrolyzed indium tin oxide (ITO) coated glasses. The chemical, morphological, and electrochemical behavior of MIP, non-imprinted polymer (NIP), and PPy-TMAO were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and electrochemical techniques. The detection response was recorded using differential pulse voltammetry (DPV), which revealed a decrease in the peak current with the increase in concentration of TMAO. The MIP sensor showed a dynamic detection range of 1–15 ppm with a sensitivity of 2.47 µA mL ppm−1 cm−2. The developed sensor is easy to construct and operate and is also highly selective to detect TMAO in body fluids such as urine. The present research provides a basis for innovative strategies to develop sensors based on MIP to detect other metabolites derived from gut microbiota that are implicated in human health and diseases.

Abstract P062: Trimethylamine N-oxide Impairs Endothelium-dependent Relaxation In Aorta Of Non-diabetic And Diabetic Mice

Trimethylamine N-oxide (TMAO) is a gut microbiota metabolite related with cardiovascular diseases. Circulating levels of TMAO are increased in diabetes and TMAO is associated with the development of insulin resistance. We aimed to investigate if TMAO impairs vascular reactivity in diabetes. The aorta of 16-week old male diabetic (db/db) and db/+ mice were mounted in a myograph and concentration-response curves to acetylcholine (ACh; 1 nM - 10 μM), sodium nitroprusside (SNP; 0.1 nM - 1 μM) and phenylephrine (PE; 1 nM - 10 μM) were obtained in the presence of TMAO (100 mM, 30 min), repeated in the presence of the antioxidant tempol (10 μM), the NLRP3 inhibitor (MCC950, 1 μM) or IL-1β receptor antagonist (IL1RA, 20 ng/mL). TMAO did not affect the contraction of PE or relaxation to SNP in aorta of db/db or db/+ mice, but decreased the potency of ACh in the aorta of db/+ (pEC 50 : 7.96 + 0.22 vs 7.25 + 0.16, lean or TMAO, respectively) and db/db (pEC 50 : 7.91 + 0.10 vs 7.17 + 0.09, db/db vs TMAO, respectively). In db/+ mice, tempol improved the potency of ACh (pEC 50 - TMAO: 7.05 + 0.21 vs TMAO + tempol 7.42 + 0.15) while further impairment in the relaxation to ACh was seen in the presence of MCC950 or IL1RA. In conclusion, TMAO impairs endothelium-dependent relaxation of aorta through the production of reactive oxygen species.

Effects of trimethylamine N-oxide on the growth and metabolism of gut bacteria

Trimethylamine N-oxide (TMAO) is an osmolyte that enters the bloodstream directly through consumption of fish, or through microbial metabolism of it and other dietary methylamines such as carnitine and choline, producing trimethylamine (TMA). TMA produced by microbes enters the blood and is transported to the liver, where it is converted back to TMAO by flavin-containing monooxygenases before being excreted in the urine. TMAO has been shown to potentially have beneficial effects on metabolic health and the gut-brain axis at normal physiological levels, while elevated serum levels are associated with cardiometabolic disease in Western patients. Bacteria in the family Enterobacteriaceae exhibit changes in their growth and metabolome in the presence of TMAO but why this occurs is not known. Growth experiments show that a caecal isolate of Klebsiella pneumoniae, a member of the Enterobacteriaceae, experience a more rapid growth rate, when grown anaerobically in the presence of TMAO, but may require oxygen to be present to produce TMA. K. pneumoniae will be subjected to differing concentrations of TMAO and oxygen to examine the effects on the metabolites produced, the genes expressed, and the rate of growth. This will be done by analysing spent media using GC-MS, qPCR targeting genes involved in anaerobic respiration, and measuring the turbidity of cultures over time. By understanding how bacteria in the gut interact with TMAO new insights can be gained as to how gut bacteria and their metabolites influence human health.

Protein Stability in TMAO and Mixed Urea-TMAO Solutions.

Osmolytes are essential for cellular function under ubiquitous osmotic stress. Trimethylamine N-oxide (TMAO) is one such osmolyte that has gained remarkable attention due to its protein-protective ability against urea. This Review aims at providing a detailed account of recent theoretical and experimental developments in characterizing the structural changes and thermodynamic stability of proteins in the presence of TMAO and urea. New vapor pressure osmometry and molecular dynamics simulation results on urea-TMAO solutions are presented, and a unified molecular mechanism of TMAO counteraction of urea-induced protein denaturation is introduced. In addition, a detailed technical assessment of molecular dynamics force fields for TMAO and for urea-TMAO solutions is presented. The force field analysis highlights how many of the commonly used force field models are in fact incompatible with solvation thermodynamics and can lead to misleading conclusions. A new optimized force field for TMAO (Shea(m)) is presented, and a recently optimized force field for TMAO-urea (Netz(m)) that best reproduces experimental data is highlighted.

Trimethylamine N‐Oxide (TMAO) Impairs Endothelium‐Dependent Relaxation in Mouse Mesenteric Resistance Artery and Aorta

Trimethylamine N‐oxide (TMAO) is a gut microbiota‐dependent metabolite produced from choline and phosphatidylcholine and it has been related to risk for cardiovascular diseases. Studies have shown that increased levels of TMAO are associated with heart attack, stroke and heart failure. This study tested the hypothesis that TMAO has a deleterious effect on the vascular beds leading to vascular dysfunction. Sixteen‐week old male lean mice were euthanized and the aorta and mesenteric resistance arteries (MRA) were mounted in a wire myograph. Concentration response curves to acetylcholine (ACh; 0.001 – 30 μM), A23187 (0.1 – 10 μM) and sodium nitroprusside (SNP; 0.001 – 1 μM) were performed in the absence or in the presence of TMAO (100 μM – incubated for 30 min) in order to evaluate endothelium dependent and independent‐relaxation of the aorta and MRA. In the aorta, TMAO decreased the potency of ACh (pEC50: 7.96 ± 0.22 versus 7.25 ± 0.16) and EMAX to ACh (89 ± 06% versus 63 ± 4%). Similarly, potency to ACh was decreased by TMAO in the MRA (pEC50: 7.52 ± 0.43 versus 6.01 ± 0.15, with vs. without TMAO, respectively). TMAO did not alter endothelium‐dependent relaxation induced by the calcium ionophore A23817 in the MRA, but completely abolished the relaxation in the aorta. Endothelium‐independent relaxation elicited by SNP was not affected by incubation with TMAO in aortic rings, whereas it caused a significant decrease in the maximal response to SNP in the MRA. In conclusion, TMAO leads to endothelial dysfunction in aorta and MRA and impaired smooth muscle relaxation of the MRA which might contribute to the development of cardiovascular diseases.Support or Funding InformationNIH: NL13604

Role of TMAO, a Gut Metabolite, on Proline‐Rich Proteins

Osmoprotectants, a group of low molecular weight compounds, are accumulated in various cells to protect organism from various stresses. Most of the osmoprotectants (osmolytes) are known to stabilize proteins by assisting them in proper folding into the native conformation. However, Trimethylamine N‐oxide (TMAO), a human gut metabolite, is one such osmolyte that is believed to have both stabilizing and destabilizing effect on different proteins. Till date the destabilizing effect of TMAO has been confined only to the fast‐folding proteins. We, therefore, investigated the destabilizing effect of TMAO on folding pattern of various slow‐folding proteins.MethodologyFolding pattern of chosen proteins in absence and presence of TMAO were studied using enzyme kinetic assays. Different spectroscopic probes were further used to measure effect on native and refolded state structure. Light Scattering measurements and electron micrographs were obtained using Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) respectively to measure aggregation status of different protein species.ResultsProtein stability and enzyme activity studies revealed TMAO acts as a denaturant. Among all osmolytes this effect was found to be unique to TMAO only. No recovery of the enzyme activity of refolded protein even after prolonged incubation without formation of aggregates confirms TMAO interfere with refolding of proteins with high number of proline residues. Structural studies revealed refolding of protein to a compact denatured state. Changed behavior of protein from denatured to native state was observed upon addition of Peptidyl‐Prolyl Isomerase (PPIase).ConclusionWe discovered that TMAO fails to stabilize or refold the slow folding proteins. Furthermore, we showed that in proteins with high number of proline residues, proline cis‐trans isomerization becomes the rate limiting step in protein folding process, thus resulting into slow folding of proteins. Recovery of native like activity of protein in presence of PPIase indicate that TMAO might have interfered cis‐trans conversion generating a non‐native protein species. This study highlights the potential role of TMAO in protein folding and associated pathological consequences.

Distinct influence of trimethylamine N-oxide and high hydrostatic pressure on community structure and culturable deep-sea bacteria

Trimethylamine N-oxide (TMAO) is one of the most important nutrients for bacteria in the deep-sea environment and is capable of improving pressure tolerance of certain bacterial strains. To assess the impact of TMAO on marine microorganisms, especially those dwelling in the deep-sea environment, we analyzed the bacterial community structure of deep-sea sediments after incubated under different conditions. Enrichments at 50 MPa and 0.1 MPa revealed that TMAO imposed a greater influence on bacterial diversity and community composition at atmospheric pressure condition than that under high hydrostatic pressure (HHP). We found that pressure was the primary factor that determines the bacterial community. Meanwhile, in total, 238 bacterial strains were isolated from the enrichments, including 112 strains affiliated to 16 genera of 4 phyla from the Yap Trench and 126 strains affiliated to 11 genera of 2 phyla from the Mariana Trench. Treatment of HHP reduced both abundance and diversity of isolates, while the presence of TMAO mainly affected the diversity of isolates obtained. In addition, certain genera were isolated only when TMAO was supplemented. Taken together, we demonstrated that pressure primarily defines the bacterial community and culturable bacterial isolates. Furthermore, we showed for the first time that TMAO had distinct influences on bacterial community depending on the pressure condition. The results enriched the understanding of the significance of TMAO in bacterial adaptation to the deep-sea environment.

Abstract P173: Trimethylamine N-Oxide (TMAO) Impairs Endothelium-Dependent Relaxation of the Mesenteric Artery in Lean and Db/db Mice

Trimethylamine N-oxide (TMAO) is a gut microbiota-dependent metabolite produced from choline and phosphatidylcholine and it has recently been related to risk for cardiovascular diseases. This study tests the hypothesis that TMAO would contribute to impaired endothelium dependent relaxation in db/db mice. Sixteen week old male lean and db/db mice were euthanized and the small mesenteric arteries (SMA) were mounted in a wire myograph. Concentration response curves to phenylephrine (PE; 1 nM- 30 μM) and acetylcholine (ACh; 1 nM- 30 μM) were performed in the absence or in the presence of TMAO (100 μM - incubated for 30 min) in order to evaluate contraction and endothelium dependent relaxation of the SMA. PE induced similar contractions of the SMA in lean and db/db mice and neither the potency nor the maximal response (E MAX ) to PE was affected in the presence of TMAO in both strains. On the other hand, there was a decrease in the potency (pEC 50 : 7.61 ± 0.19 lean; 6.67 ± 0.25 db/db; n=6) and E MAX (9.73 ± 0.43 mN lean; 8.41 ± 0.62 mN db/db; n=6) to ACh in the SMA of db/db mice. TMAO led to a decrease the E MAX to ACh in both strains (7.52 ± 0.43 and 5.46 ± 0.46, for lean and db/db respectively) and the potency of ACh only in lean mice (pEC 50 : 6.01 ± 0.15 and 6.13 ± 0.24, for lean and db/db respectively) when compared to the respective control in the absence of TMAO. In conclusion, TMAO may contribute to the development of cardiovascular complications by impairing the vascular relaxation.

Cosolvent and Crowding Effects on the Temperature- and Pressure-Dependent Dissociation Process of the α/β-Tubulin Heterodimer.

Tubulin is one of the main components of the cytoskeleton of eukaryotic cells. The formation of microtubules depends strongly on environmental and solution conditions, and has been found to be among the most pressure sensitive processes in vivo. We explored the effects of different types of cosolvents, such as trimethylamine-N-oxide (TMAO), sucrose and urea, and crowding agents to mimic cell-like conditions, on the temperature and pressure stability of the building block of microtubules, i. e. the α/β-tubulin heterodimer. To this end, fluorescence and FTIR spectroscopy, differential scanning and pressure perturbation calorimetry as well as fluorescence anisotropy and correlation spectroscopies were applied. The pressure and temperature of dissociation of α/β-tubulin as well as the underlying thermodynamic parameters upon dissociation, such as volume and enthalpy changes, have been determined for the different solution conditions. The temperature and pressure of dissociation of the α/β-tubulin heterodimer and hence its stability increases dramatically in the presence of TMAO and the nanocrowder sucrose. We show that by adjusting the levels of compatible cosolutes and crowders, cells are able to withstand deteriorating effects of pressure even up to the kbar-range.

Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota

BackgroundThe dietary methylamines choline, carnitine, and phosphatidylcholine are used by the gut microbiota to produce a range of metabolites, including trimethylamine (TMA). However, little is known about the use of trimethylamine N-oxide (TMAO) by this consortium of microbes.ResultsA feeding study using deuterated TMAO in C57BL6/J mice demonstrated microbial conversion of TMAO to TMA, with uptake of TMA into the bloodstream and its conversion to TMAO. Microbial activity necessary to convert TMAO to TMA was suppressed in antibiotic-treated mice, with deuterated TMAO being taken up directly into the bloodstream. In batch-culture fermentation systems inoculated with human faeces, growth of Enterobacteriaceae was stimulated in the presence of TMAO. Human-derived faecal and caecal bacteria (n = 66 isolates) were screened on solid and liquid media for their ability to use TMAO, with metabolites in spent media analysed by 1H-NMR. As with the in vitro fermentation experiments, TMAO stimulated the growth of Enterobacteriaceae; these bacteria produced most TMA from TMAO. Caecal/small intestinal isolates of Escherichia coli produced more TMA from TMAO than their faecal counterparts. Lactic acid bacteria produced increased amounts of lactate when grown in the presence of TMAO but did not produce large amounts of TMA. Clostridia (sensu stricto), bifidobacteria, and coriobacteria were significantly correlated with TMA production in the mixed fermentation system but did not produce notable quantities of TMA from TMAO in pure culture.ConclusionsReduction of TMAO by the gut microbiota (predominantly Enterobacteriaceae) to TMA followed by host uptake of TMA into the bloodstream from the intestine and its conversion back to TMAO by host hepatic enzymes is an example of metabolic retroconversion. TMAO influences microbial metabolism depending on isolation source and taxon of gut bacterium. Correlation of metabolomic and abundance data from mixed microbiota fermentation systems did not give a true picture of which members of the gut microbiota were responsible for converting TMAO to TMA; only by supplementing the study with pure culture work and additional metabolomics was it possible to increase our understanding of TMAO bioconversions by the human gut microbiota.