William R. Wikoff

Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites

Although it has long been recognized that the enteric community of bacteria that inhabit the human distal intestinal track broadly impacts human health, the biochemical details that underlie these effects remain largely undefined. Here, we report a broad MS-based metabolomics study that demonstrates a surprisingly large effect of the gut “microbiome” on mammalian blood metabolites. Plasma extracts from germ-free mice were compared with samples from conventional (conv) animals by using various MS-based methods. Hundreds of features were detected in only 1 sample set, with the majority of these being unique to the conv animals, whereas ≈10% of all features observed in both sample sets showed significant changes in their relative signal intensity. Amino acid metabolites were particularly affected. For example, the bacterial-mediated production of bioactive indole-containing metabolites derived from tryptophan such as indoxyl sulfate and the antioxidant indole-3-propionic acid (IPA) was impacted. Production of IPA was shown to be completely dependent on the presence of gut microflora and could be established by colonization with the bacterium Clostridium sporogenes. Multiple organic acids containing phenyl groups were also greatly increased in the presence of gut microbes. A broad, drug-like phase II metabolic response of the host to metabolites generated by the microbiome was observed, suggesting that the gut microflora has a direct impact on the drug metabolism capacity of the host. Together, these results suggest a significant interplay between bacterial and mammalian metabolism.

Metabolomic analysis of the cerebrospinal fluid reveals changes in phospholipase expression in the CNS of SIV-infected macaques

HIV infiltrates the CNS soon after an individual has become infected with the virus, and can cause dementia and encephalitis in late-stage disease. Here, a global metabolomics approach was used to find and identify metabolites differentially regulated in the cerebrospinal fluid (CSF) of rhesus macaques with SIV-induced CNS disease, as we hypothesized that this might provide biomarkers of virus-induced CNS damage. The screening platform used a non-targeted, mass-based metabolomics approach beginning with capillary reverse phase chromatography and electrospray ionization with accurate mass determination, followed by novel, nonlinear data alignment and online database screening to identify metabolites. CSF was compared before and after viral infection. Significant changes in the metabolome specific to SIV-induced encephalitis were observed. Metabolites that were increased during infection-induced encephalitis included carnitine, acyl-carnitines, fatty acids, and phospholipid molecules. The elevation in free fatty acids and lysophospholipids correlated with increased expression of specific phospholipases in the brains of animals with encephalitis. One of these, a phospholipase A2 isoenzyme, is capable of releasing a number of the fatty acids identified. It was expressed in different areas of the brain in conjunction with glial activation, rather than linked to regions of SIV infection and inflammation, indicating widespread alterations in infected brains. The identification of specific metabolites as well as mechanisms of their increase illustrates the potential of mass-based metabolomics to address problems in CNS biochemistry and neurovirology, as well as neurodegenerative diseases.

Variability Analysis of Human Plasma and Cerebral Spinal Fluid Reveals Statistical Significance of Changes in Mass Spectrometry-Based Metabolomics Data

Analytical and biological variability are issues of central importance to human metabolomics studies. Here both types of variation are examined in human plasma and cerebrospinal fluid (CSF) using a global liquid chromatography/mass spectrometry (LC/MS) metabolomics strategy. The platform shows small analytical variation with a median coefficient of variation (CV) of 15-16% for both plasma and CSF sample matrixes when the integrated area of each peak in the mass spectra is considered. Analysis of biological variation shows that human CSF has a median CV of 35% and plasma has a median CV of 46%. To understand the difference in CV between the biofluids, we compared plasma and CSF independently obtained from different healthy humans. Additionally, we analyzed another group of patients from whom we compared matched CSF and plasma (plasma and CSF obtained from the same human subject). A similar number of features was observed in both biofluids, although the majority of features appeared with greater intensity in plasma. More than a dozen metabolites shared between the human CSF and plasma metabolomes were identified based on accurate mass measurements, retention times, and MS/MS spectra. The fold change in these metabolites was consistent with the median biological CV determined for all peaks. The measured median biological CV together with analysis of intragroup variation of healthy individuals suggests that fold changes above 2 in metabolomics studies investigating plasma or CSF are statistically relevant with respect to the inherent variability of a healthy control group. These data demonstrate the reproducibility of the global metabolomics platform using LC/MS and reveal the robustness of the approach for biomarker discovery.

Untargeted metabolomics identifies enterobiome metabolites and putative uremic toxins as substrates of organic anion transporter 1 (Oat1).

Untargeted metabolomics on the plasma and urine from wild-type and organic anion transporter-1 (Oat1/Slc22a6) knockout mice identified a number of physiologically important metabolites, including several not previously linked to Oat1-mediated transport. Several, such as indoxyl sulfate, derive from Phase II metabolism of enteric gut precursors and accumulate in chronic kidney disease (CKD). Other compounds included vitamins (pantothenic acid, 4-pyridoxic acid), urate, and metabolites in the tryptophan and nucleoside pathways. Three metabolites, indoxyl sulfate, kynurenine, and xanthurenic acid, were elevated in the plasma and interacted strongly and directly with Oat1 in vitro with IC50 of 18, 12, and 50 μM, respectively. A pharmacophore model based on several identified Oat1 substrates was used to screen the NCI database and candidate compounds interacting with Oat1 were validated in an in vitro assay. Together, the data suggest a complex, previously unidentified remote communication between the gut microbiome, Phase II metabolism in the liver, and elimination via Oats of the kidney, as well as indicating the importance of Oat1 in the handling of endogenous toxins associated with renal failure and uremia. The possibility that some of the compounds identified may be part of a larger remote sensing and signaling pathway is also discussed.

The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment.

BACKGROUND Members of the Parvovirus genus cause a variety of diseases in mammals, including humans. One of the major defences against viral infection is the presence of neutralizing antibodies that prevent virus particles from infecting target cells. The mechanism of neutralization is not well understood. We therefore studied the structure of canine parvovirus (CPV) complexed with the Fab fragment of a neutralizing antibody, A3B10, using image reconstruction of electron micrographs of vitrified samples, together with the already known structure of CPV from X-ray crystallographic data. RESULTS The structure of the complex of CPV with Fab A3B10 has been determined to 23 A resolution. The known CPV atomic structure was subtracted from the electron density of the complex, and the difference map was used to fit the atomic coordinates of a known Fab fragment, HyHEL-5. The long axis of each Fab molecule is oriented in a near radial direction, inclined away from the two-fold axes. The viral epitope consists of 14 amino acid residues found in loops 1, 2 and 3 on the capsid surface, which include previously identified escape mutations. CONCLUSIONS The mode of Fab binding suggests that the A3B10 neutralizing antibody cannot bind bivalently to the capsid across the two-fold axes, consistent with the observation that whole A3B10 antibody readily precipitates CPV. Since Fab A3B10 can also neutralize the virus, mechanisms of neutralization such as interference with cell attachment, cell entry, or uncoating, must be operative.

Organic Anion Transporter 3 Contributes to the Regulation of Blood Pressure

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension.

Novel Multiprotein Complexes Identified in the Hyperthermophilic Archaeon Pyrococcus furiosus by Non-denaturing Fractionation of the Native Proteome

Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 °C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing ∼80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.

Virus assembly: Imaging a molecular machine.

A recent structural study of a double-stranded DNA bacteriophage has provided remarkable new insights into the assembly of a complex virus particle and ushers in a new era in the imaging of non-icosahedral viruses.

Mobilization of pro-inflammatory lipids in obese Plscr3-deficient mice

BackgroundThe obesity epidemic has prompted the search for candidate genes capable of influencing adipose function. One such candidate, that encoding phospholipid scramblase 3 (PLSCR3), was recently identified, as genetic deletion of it led to lipid accumulation in abdominal fat pads and changes characteristic of metabolic syndrome. Because adipose tissue is increasingly recognized as an endocrine organ, capable of releasing small molecules that modulate disparate physiological processes, we examined the plasma from wild-type, Plscr1-/-, Plscr3-/- and Plscr1&3-/- mice. Using an untargeted comprehensive metabolite profiling approach coupled with targeted gene expression analyses, the perturbed biochemistry and functional redundancy of PLSCR proteins was assessed.ResultsNineteen metabolites were differentially and similarly regulated in both Plscr3-/- and Plscr1&3-/- animals, of which five were characterized from accurate mass, tandem mass spectrometry data and their correlation to the Metlin database as lysophosphatidylcholine (LPC) species enriched with C16:1, C18:1, C20:3, C20:5 and C22:5 fatty acids. No significant changes in the plasma metabolome were detected upon elimination of PLSCR1, indicating that increases in pro-inflammatory lipids are specifically associated with the obese state of Plscr3-deficient animals. Correspondingly, increases in white adipose lipogenic gene expression confirm a role for PLSCR3 in adipose lipid metabolism.ConclusionThe untargeted profiling of circulating metabolites suggests no detectable functional redundancies between PLSCR proteins; however, this approach simultaneously identified previously unrecognized lipid metabolites that suggest a novel molecular link between obesity, inflammation and the downstream consequences associated with PLSCR3-deficiency.

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is similar in architecture to bacteriophage lambda, the type member of this family, with an icosahedral capsid of triangulation number T = 7. No high-resolution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-rays. At 650 A in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. The empty Head II (mature) particles were expressed in Escherichia coli and assembled in vitro, but they have the same morphology as the mature HK97 capsid. Previously reported Head II crystals diffracting to 3.5 A resolution are examined here in detail. Although the cell dimensions suggest an orthorhombic lattice, further analysis demonstrated that the space group was monoclinic. This has been confirmed by the present study. Images were recorded on the F1 beamline at CHESS and they were processed and scaled, resulting in a data set with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 A. The cell dimensions after post-refinement were a = 580, b = 626, c = 788 A, beta = 90.0 degrees. From the particle dimensions determined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even reflections along the 0k0 lattice line indicate that the space group is P21. The rotation function was used to determine the orientation of the particles in the unit cell and to confirm the space group. An icosahedral twofold axis is approximately, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to the b axis, is rotated 18 degrees away from the a axis. The centers of the two particles must be positioned close to the minimum-energy packing arrangement for spheres, which places one particle at ((1/4), 0, (1/4)) and the other particle at ((3/4), (1/2), (3/4)). The particle position and orientation were confirmed by calculating a Patterson function. The particles interact closely along icosahedral threefold axes, which occurs both along the crystallographic a axis and along the b axis. The particle dimensions derived from this packing arrangement agree well with those determined by cryo-EM and image reconstruction. The cryo-EM reconstruction will be used as a model to initiate phase determination; structure determination at 7 A is under way.