Bio21 - Research Publications
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ItemNo Preview AvailableRemodeling of Carbon Metabolism during Sulfoglycolysis in Escherichia coli(AMER SOC MICROBIOLOGY, 2023-02-28)Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E. coli cells was undertaken to identify the metabolic consequences of the switch from glycolysis to sulfoglycolysis. Sulfoglycolysis was associated with the diversion of triose phosphates (triose-P) to synthesize sugar phosphates (gluconeogenesis) and an unexpected accumulation of trehalose and glycogen storage carbohydrates. Sulfoglycolysis was also associated with global changes in central carbon metabolism, as indicated by the changes in the levels of intermediates in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), polyamine metabolism, pyrimidine metabolism, and many amino acid metabolic pathways. Upon entry into stationary phase and the depletion of SQ, E. coli cells utilize their glycogen, indicating a reversal of metabolic fluxes to allow glycolytic metabolism. IMPORTANCE The sulfosugar sulfoquinovose is estimated to be produced on a scale of 10 billion metric tons per annum, making it a major organosulfur species in the biosulfur cycle. The microbial degradation of sulfoquinovose through sulfoglycolysis allows the utilization of its carbon content and contributes to the biomineralization of its sulfur. However, the metabolic consequences of microbial growth on sulfoquinovose are unclear. We use metabolomics to identify the metabolic adaptations that Escherichia coli undergoes when grown on sulfoquinovose versus glucose. This revealed the increased flux into storage carbohydrates through gluconeogenesis and the reduced flux of carbon into the TCA cycle and downstream metabolism. These changes are relieved upon entry into stationary phase and reversion to glycolytic metabolism. This work provides new insights into the metabolic consequences of microbial growth on an abundant sulfosugar.
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ItemType I interferon antagonism of the JMJD3-IRF4 pathway modulates macrophage activation and polarization(CELL PRESS, 2022-04-19)Metabolic adaptations can directly influence the scope and scale of macrophage activation and polarization. Here we explore the impact of type I interferon (IFNβ) on macrophage metabolism and its broader impact on cytokine signaling pathways. We find that IFNβ simultaneously increased the expression of immune-responsive gene 1 and itaconate production while inhibiting isocitrate dehydrogenase activity and restricting α-ketoglutarate accumulation. IFNβ also increased the flux of glutamine-derived carbon into the tricarboxylic acid cycle to boost succinate levels. Combined, we identify that IFNβ controls the cellular α-ketoglutarate/succinate ratio. We show that by lowering the α-ketoglutarate/succinate ratio, IFNβ potently blocks the JMJD3-IRF4-dependent pathway in GM-CSF and IL-4 activated macrophages. The suppressive effects of IFNβ on JMJD3-IRF4-dependent responses, including M2 polarization and GM-CSF-induced inflammatory pain, were reversed by supplementation with α-ketoglutarate. These results reveal that IFNβ modulates macrophage activation and polarization through control of the cellular α-ketoglutarate/succinate ratio.
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ItemLipidomic Profiling of Adipose Tissue Reveals an Inflammatory Signature in Cancer-Related and Primary Lymphedema(PUBLIC LIBRARY SCIENCE, 2016-05-16)Cancer-related and primary lymphedema (LE) are associated with the production of adipose tissue (AT). Nothing is known, however, about the lipid-based molecules that comprise LE AT. We therefore analyzed lipid molecules in lipoaspirates and serum obtained from LE patients, and compared them to lipoaspirates from cosmetic surgery patients and healthy control cohort serum. LE patient serum analysis demonstrated that triglycerides, HDL- and LDL-cholesterol and lipid transport molecules remained within the normal range, with no alterations in individual fatty acids. The lipidomic analysis also identified 275 lipid-based molecules, including triacylglycerides, diacylglycerides, fatty acids and phospholipids in AT oil and fat. Although the majority of lipid molecules were present in a similar abundance in LE and non-LE samples, there were several small changes: increased C20:5-containing triacylglycerides, reduced C10:0 caprinic and C24:1 nervonic acids. LE AT oil also contained a signature of increased cyclopropane-type fatty acids and inflammatory mediators arachidonic acid and ceramides. Interestingly C20:5 and C22:6 omega-3-type lipids are increased in LE AT, correlating with LE years. Hence, LE AT has a normal lipid profile containing a signature of inflammation and omega-3-lipids. It remains unclear, however, whether these differences reflect a small-scale global metabolic disturbance or effects within localised inflammatory foci.
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ItemReversing diet-induced metabolic dysregulation by diet switching leads to altered hepatic de novo lipogenesis and glycerolipid synthesis(NATURE PORTFOLIO, 2016-06-07)In humans, low-energy diets rapidly reduce hepatic fat and improve/normalise glycemic control. Due to difficulties in obtaining human liver, little is known about changes to the lipid species and pathway fluxes that occur under these conditions. Using a combination of stable isotope, and targeted metabolomic approaches we investigated the acute (7-9 days) hepatic effects of switching high-fat high-sucrose diet (HFD) fed obese mice back to a chow diet. Upon the switch, energy intake was reduced, resulting in reductions of fat mass and hepatic triacyl- and diacylglycerol. However, these parameters were still elevated compared to chow fed mice, thus representing an intermediate phenotype. Nonetheless, glucose intolerance and hyperinsulinemia were completely normalized. The diet reversal resulted in marked reductions in hepatic de novo lipogenesis when compared to the chow and HFD groups. Compared with HFD, glycerolipid synthesis was reduced in the reversal animals, however it remained elevated above that of chow controls, indicating that despite experiencing a net loss in lipid stores, the liver was still actively esterifying available fatty acids at rates higher than that in chow control mice. This effect likely promotes the re-esterification of excess free fatty acids released from the breakdown of adipose depots during the weight loss period.
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ItemTalaromyces marneffei simA Encodes a Fungal Cytochrome P450 Essential for Survival in Macrophages(AMER SOC MICROBIOLOGY, 2018)Fungi are adept at occupying specific environmental niches and often exploit numerous secondary metabolites generated by the cytochrome P450 (CYP) monoxygenases. This report describes the characterization of a yeast-specific CYP encoded by simA ("survival in macrophages"). Deletion of simA does not affect yeast growth at 37°C in vitro but is essential for yeast cell production during macrophage infection. The ΔsimA strain exhibits reduced conidial germination and intracellular growth of yeast in macrophages, suggesting that the enzymatic product of SimA is required for normal fungal growth in vivo. Intracellular ΔsimA yeast cells exhibit cell wall defects, and metabolomic and chemical sensitivity data suggest that SimA may promote chitin synthesis or deposition in vitro. In vivo, ΔsimA yeast cells subsequently lyse and are degraded, suggesting that SimA may increase resistance to and/or suppress host cell biocidal effectors. The results suggest that simA synthesizes a secondary metabolite that allows T. marneffei to occupy the specific intracellular environmental niche within the macrophage. IMPORTANCE This study in a dimorphic fungal pathogen uncovered a role for a yeast-specific cytochrome P450 (CYP)-encoding gene in the ability of T. marneffei to grow as yeast cells within the host macrophages. This report will inspire further research into the role of CYPs and secondary metabolite synthesis during fungal pathogenic growth.
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ItemMicrobe-Metabolite Associations Linked to the Rebounding Murine Gut Microbiome Postcolonization with Vancomycin-Resistant Enterococcus faecium(AMER SOC MICROBIOLOGY, 2020-08-18)Vancomycin-resistant Enterococcus faecium (VREfm) is an emerging antibiotic-resistant pathogen. Strain-level investigations are beginning to reveal the molecular mechanisms used by VREfm to colonize regions of the human bowel. However, the role of commensal bacteria during VREfm colonization, in particular following antibiotic treatment, remains largely unknown. We employed amplicon 16S rRNA gene sequencing and metabolomics in a murine model system to try and investigate functional roles of the gut microbiome during VREfm colonization. First-order taxonomic shifts between Bacteroidetes and Tenericutes within the gut microbial community composition were detected both in response to pretreatment using ceftriaxone and to subsequent VREfm challenge. Using neural networking approaches to find cooccurrence profiles of bacteria and metabolites, we detected key metabolome features associated with butyric acid during and after VREfm colonization. These metabolite features were associated with Bacteroides, indicative of a transition toward a preantibiotic naive microbiome. This study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in response to colonization with VREfm. Our results offer insights toward identifying potential nonantibiotic alternatives to eliminate VREfm through metabolic reengineering to preferentially select for Bacteroides IMPORTANCE This study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions between commensal gut bacteria and a specific pathogen. Knowledge from this research will inform gut microbiome engineering strategies, with the aim of translating observations from animal models to human-relevant therapeutic applications.
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ItemMus musculus deficient for secretory antibodies show delayed growth with an altered urinary metabolome(SPRINGER, 2019-04-03)BACKGROUND: The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial barriers by transporting polymeric antibodies and antigens through the epithelial mucosa into the lumen. In this study, we examined the role of pIgR in maintaining gut barrier integrity, which is important for the normal development in mice. METHODS: Cohorts of pIgR-/- mice and their wildtype controls were housed under Specific Pathogen Free (SPF) conditions and monitored for weight gain as an indicator of development over time. The general physiology of the gastrointestinal tract was analysed using immunohistochemistry in young (8-12 weeks of age) and aged mice (up to 18 months of age), and the observed immunopathology in pIgR-/- mice was further characterised using flow cytometry. Urinary metabolites were analysed using gas chromatography-mass spectrometry (GC-MS), which revealed changes in metabolites that correlated with age-related increase in gut permeability in pIgR-/- mice. RESULTS: We observed that pIgR-/- mice exhibited delayed growth, and this phenomenon is associated with low-grade gut inflammation that increased with ageing. The gross intraepithelial lymphocytic (IEL) infiltration characteristic of pIgR-/- mice was redefined as CD8α+αβ+ T cells, the majority of which expressed high levels of CD103 and CD69 consistent with tissue resident memory T cells (TRM). Comparison of the urinary metabolome between pIgR-/- and wild-type mice revealed key changes in urinary biomarkers fucose, glycine and Vitamin B5, suggestive of altered mucosal permeability. A significant increase in gut permeability was confirmed by analysing the site-specific uptake of sugar probes in different parts of the intestine. CONCLUSION: Our data show that loss of the secretory antibody system in mice results in enhanced accumulation of inflammatory IELs in the gut, which likely reflects ongoing inflammation in reaction to gut microbiota or food antigens, leading to delayed growth in pIgR-/- mice. We demonstrate that this leads to the presence of a unique urinary metabolome profile, which may provide a biomarker for altered gut permeability.
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ItemNLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease(NATURE PUBLISHING GROUP, 2018-09-13)Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.
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ItemCircadian and wake-dependent changes in human plasma polar metabolites during prolonged wakefulness: A preliminary analysis(NATURE PUBLISHING GROUP, 2019-03-14)Establishing circadian and wake-dependent changes in the human metabolome are critical for understanding and treating human diseases due to circadian misalignment or extended wake. Here, we assessed endogenous circadian rhythms and wake-dependent changes in plasma metabolites in 13 participants (4 females) studied during 40-hours of wakefulness. Four-hourly plasma samples were analyzed by hydrophilic interaction liquid chromatography (HILIC)-LC-MS for 1,740 metabolite signals. Group-averaged (relative to DLMO) and individual participant metabolite profiles were fitted with a combined cosinor and linear regression model. In group-level analyses, 22% of metabolites were rhythmic and 8% were linear, whereas in individual-level analyses, 14% of profiles were rhythmic and 4% were linear. We observed metabolites that were significant at the group-level but not significant in a single individual, and metabolites that were significant in approximately half of individuals but not group-significant. Of the group-rhythmic and group-linear metabolites, only 7% and 12% were also significantly rhythmic or linear, respectively, in ≥50% of participants. Owing to large inter-individual variation in rhythm timing and the magnitude and direction of linear change, acrophase and slope estimates also differed between group- and individual-level analyses. These preliminary findings have important implications for biomarker development and understanding of sleep and circadian regulation of metabolism.
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ItemFunction of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjaerg syndrome(ELIFE SCIENCES PUBLICATIONS LTD, 2019-11-04)Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.