Modulation of acyl-carnitines, the broad mechanism behind Wolbachia-mediated inhibition of medically important flaviviruses in Aedes aegypti.

Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

An integrated metagenomics and metabolomics approach implicates the microbiota-gut-brain axis in the pathogenesis of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors. Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors. Using shotgun sequencing, we investigated the gut microbiome composition in the R6/1 transgenic mouse model of HD from 4 to 12 weeks of age (early adolescent through to adult stages). Targeted metabolomics was also performed on the blood plasma of these mice (n = 9 per group) at 12 weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile. RESULTS: Modelled time profiles of each species, KEGG Orthologs and bacterial genes, revealed heightened volatility in the R6/1 mice, indicating potential early effects of the HD mutation in the gut. In addition to gut dysbiosis in R6/1 mice at 12 weeks of age, gut microbiome function was perturbed. In particular, the butanoate metabolism pathway was elevated, suggesting increased production of the protective SCFA, butyrate, in the gut. No significant alterations were found in the plasma butyrate and propionate levels in the R6/1 mice at 12 weeks of age. The statistical integration of the metagenomics and metabolomics unraveled several Bacteroides species that were negatively correlated with ATP and pipecolic acid in the plasma. CONCLUSIONS: The present study revealed the instability of the HD gut microbiome during the pre-motor symptomatic stage of the disease which may have dire consequences on the host's health. Perturbation of the HD gut microbiome function prior to significant cognitive and motor dysfunction suggest the potential role of the gut in modulating the pathogenesis of HD, potentially via specific altered plasma metabolites which mediate gut-brain signaling.

Using metabolomics to assess the sub-lethal effects of zinc and boscalid on an estuarine polychaete worm over time.

INTRODUCTION: Zinc is a heavy metal commonly detected in urban estuaries around Australia. Boscalid is a fungicide found in estuaries, both in water and sediment, it enters the system predominantly through agricultural run-off. Zinc is persistent while boscalid breaks down, with a half-life of 108 days. Both contaminants are widely distributed and their effects on ecosystems are not well understood. OBJECTIVES: The aim of this study was to determine the metabolite changes in Simplisetia aequisetis (an estuarine polychaete) following laboratory exposure to a sub-lethal concentration of zinc or boscalid over a 2-week period. METHODS: Individuals were collected at six time points over a 2-week period. Whole polychaete metabolites were extracted and quantified using a multi-platform approach. Polar metabolites were detected using a semi-targeted GC-MS analysis and amine containing compounds were analysed using a targeted LC-MS analysis. Total lipid energy content was also analysed for Simplisetia aequisetis. RESULTS: The pathways that responded to zinc and boscalid exposure were alanine, aspartate and glutamate metabolism (AAG); glycine, serine and threonine metabolism (GST) and metabolites associated with the tricarboxylic acid cycle (TCA). Results showed that changes in total abundance of some metabolites could be detected as early as 24-h exposure. Changes were detected in the metabolites before commonly used total lipid energy assays identified effects. CONCLUSION: A multi-platform approach provided a holistic overview of the metabolomic response to contaminants in polychaetes. This approach shows promise to be used in biomonitoring programs to provide early diagnostic indicators of contamination and exposure.

Review of recent developments in GC-MS approaches to metabolomics-based research.

BACKGROUND: Metabolomics aims to identify the changes in endogenous metabolites of biological systems in response to intrinsic and extrinsic factors. This is accomplished through untargeted, semi-targeted and targeted based approaches. Untargeted and semi-targeted methods are typically applied in hypothesis-generating investigations (aimed at measuring as many metabolites as possible), while targeted approaches analyze a relatively smaller subset of biochemically important and relevant metabolites. Regardless of approach, it is well recognized amongst the metabolomics community that gas chromatography-mass spectrometry (GC-MS) is one of the most efficient, reproducible and well used analytical platforms for metabolomics research. This is due to the robust, reproducible and selective nature of the technique, as well as the large number of well-established libraries of both commercial and 'in house' metabolite databases available. AIM OF REVIEW: This review provides an overview of developments in GC-MS based metabolomics applications, with a focus on sample preparation and preservation techniques. A number of chemical derivatization (in-time, in-liner, offline and microwave assisted) techniques are also discussed. Electron impact ionization and a summary of alternate mass analyzers are highlighted, along with a number of recently reported new GC columns suited for metabolomics. Lastly, multidimensional GC-MS and its application in environmental and biomedical research is presented, along with the importance of bioinformatics. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is to both highlight and provide an update on GC-MS analytical techniques that are common in metabolomics studies. Specific emphasis is given to the key steps within the GC-MS workflow that those new to this field need to be aware of and the common pitfalls that should be looked out for when starting in this area.

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood.

BACKGROUND: Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing. METHOD: Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy. RESULTS: Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased. CONCLUSION: Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris.

Protein mannosyltransferases (PMTs) catalyze the O-mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 &7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O-mannosylated. In this study, we attempted to prevent O-mannosylation of insulin precursor in vivo, through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O-mannosylation of the insulin precursor. The genes encoding PMT1, 4, 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16-28% reductions in the O-mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O-mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O-mannosylation.

Microbe-Metabolite Associations Linked to the Rebounding Murine Gut Microbiome Postcolonization with Vancomycin-Resistant Enterococcus faecium.

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.

Metabolite Changes in an Estuarine Annelid Following Sublethal Exposure to a Mixture of Zinc and Boscalid.

Environmental pollutants such as heavy metals and fungicides pose a serious threat to waterways worldwide. Toxicological assessment of such contaminants is usually conducted using single compound exposures, as it is challenging to understand the effect of mixtures on biota using standard ecotoxicological methods; whereas complex chemical mixtures are more probable in ecosystems. This study exposed Simplisetia aequisetis (an estuarine annelid) to sublethal concentrations of a metal (zinc) and a fungicide (boscalid), both singly and as a mixture, for two weeks. Metabolomic analysis via gas and liquid chromatography-mass spectrometry was used to measure the stress response(s) of the organism following exposure. A total of 75 metabolites, including compounds contributing to the tricarboxylic acid cycle, the urea cycle, and a number of other pathways, were identified and quantified. The multiplatform approach identified distinct metabolomic responses to each compound that differed depending on whether the substance was presented singly or as a mixture, indicating a possible antagonistic effect. The study demonstrates that metabolomics is able to elucidate the effects and mode of action of contaminants and can identify possible outcomes faster than standard ecotoxicological endpoints, such as growth and reproduction. Metabolomics therefore has a possible future role in biomonitoring and ecosystem health assessments.

NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease.

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.