RESUMO
Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.
Assuntos
Escherichia coli , Lipopolissacarídeos , Lipopolissacarídeos/química , Escherichia coli/química , Bacteroides fragilis/química , Elétrons , Espectrometria de Massas em TandemRESUMO
Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and that palmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD (D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.
Assuntos
Gasderminas , Lipoilação , Proteínas de Ligação a Fosfato , Espécies Reativas de Oxigênio , Animais , Feminino , Humanos , Masculino , Camundongos , Aciltransferases/metabolismo , Microscopia Crioeletrônica , Cisteína/metabolismo , Gasderminas/química , Gasderminas/metabolismo , Inflamassomos/metabolismo , Lipossomos/metabolismo , Lipossomos/química , Mitocôndrias/metabolismo , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Células THP-1RESUMO
The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites1-3. Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine4. Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties5. However, little is known about how this diet-microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4+ intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4+CD8αα+ IELs in gnotobiotic mice. Restoration of CLAs increases CD4+CD8αα+ IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4+CD8αα+ IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4+ T cells that were CD4+CD8αα+.
Assuntos
Ácidos Graxos , Microbioma Gastrointestinal , Linfócitos Intraepiteliais , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Isomerismo , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Lipólise , Ácido Linoleico/metabolismo , Imunidade nas MucosasRESUMO
Bioactive metabolites produced by symbiotic microbiota causally impact host health and disease, nonetheless, incomplete functional annotation of genes as well as complexities and dynamic nature of microbiota make understanding species-level contribution in production and actions difficult. Alpha-galactosylceramides produced by Bacteroides fragilis (BfaGC) are one of the first modulators of colonic immune development, but biosynthetic pathways and the significance of the single species in the symbiont community still remained elusive. To address these questions at the microbiota level, we have investigated the lipidomic profiles of prominent gut symbionts and the metagenome-level landscape of responsible gene signatures in the human gut. We first elucidated the chemical diversity of sphingolipid biosynthesis pathways of major bacterial species. In addition to commonly shared ceramide backbone synthases showing two distinct intermediates, alpha-galactosyltransferase (agcT), the necessary and sufficient component for BfaGC production and host colonic type I natural killer T (NKT) cell regulation by B. fragilis, was characterized by forward-genetics based targeted metabolomic screenings. Phylogenetic analysis of agcT in human gut symbionts revealed that only a few ceramide producers have agcT and hence can produce aGCs, on the other hand, structurally conserved homologues of agcT are widely distributed among species lacking ceramides. Among them, alpha-glucosyl-diacylglycerol(aGlcDAG)-producing glycosyltransferases with conserved GT4-GT1 domains are one of the most prominent homologs in gut microbiota, represented by Enterococcus bgsB . Of interest, aGlcDAGs produced by bgsB can antagonize BfaGC-mediated activation of NKT cells, showing the opposite, lipid structure-specific actions to regulate host immune responses. Further metagenomic analysis of multiple human cohorts uncovered that the agcT gene signature is almost exclusively contributed by B. fragilis , regardless of age, geographical and health status, where the bgsB signature is contributed by >100 species, of which abundance of individual microbes is highly variable. Our results collectively showcase the diversities of gut microbiota producing biologically relevant metabolites in multiple layers-biosynthetic pathways, host immunomodulatory functions and microbiome-level landscapes in the host.
RESUMO
Symbiotic microbiota critically contribute to host immune homeostasis in effector cell-specific manner. For exclusion of microbial component, germ-free animals have been the gold standard method. However, total removal of the entire gut microbiota of an animal from birth significantly skews physiological development. On the other hand, removal of gut microbiota from conventional mice using oral antibiotics has its own limitations, especially lack of consistency and the requirement for long-term treatment period. Here, we introduce an improved regimen to quickly remove gut microbiota and to maintain sterility, that is well received by animals without refusal. Rapid and consistent exclusion of resident bacteria in the gut lumen revealed kinetic differences among colonic lymphocyte subsets, which cannot be observed with typical germ-free animal models. Furthermore, the proposed method distinguished the mechanism of microbiota contribution as a direct stimulus to capable effector cells and a homeostatic cue to maintain such cell types.
Assuntos
Microbioma Gastrointestinal , Microbiota , Animais , Camundongos , Microbiota/fisiologia , Colo , Bactérias/genética , Bactérias/metabolismo , HomeostaseRESUMO
Lipid phosphate phosphatases are a family of enzymes with diverse cellular metabolic functions. Phospholipid phosphatase 6 (PLPP6) is a regulator of cellular polyisoprenyl phosphates; however, its in vivo functions remain to be determined. Here, mouse PLPP6 was characterized to possess similar catalytic properties as the human enzyme. Plpp6 knockout mice (Plpp6 -/- ) were generated and displayed decreased airway allergen sensitization, pointing to a role for PLPP6 in the early events of lung allergic responses. Dendritic cell (DC) responses were investigated and endocytosis of allergen via macropinocytosis was decreased in Plpp6 -/- DCs that had lower cholesterol content. When reversed by cholesterol loading, the DC macropinocytosis defect is corrected. Adoptive transfer of Plpp6 -/- DCs to wild-type mice during sensitization was sufficient to decrease allergen-induced responses. Together, our findings have identified PLPP6 as a pivotal regulator of DC cholesterol content and macropinocytosis, cellular mechanisms that are important for pathologic responses in allergen-induced lung inflammation.
RESUMO
Sodium taurocholate cotransporting polypeptide (NTCP) involved in bile acid transport in the liver is an entry receptor of hepatitis B virus (HBV). In the present study, we introduce a mass spectrometric screening assay for targeting HBV entry inhibitors that can reduce NTCP transporter activity by employing taurocholic acid (TCA) labeled with stable isotope (2,2,4,4-d4-TCA, d4-TCA) and NTCP-overexpressing human liver cancer cell lines such as HepG2 and Huh-7. The accuracy and reliability of the proposed mass spectrometric NTCP activity assay have been validated with known HBV inhibitors including cyclosporine A (CsA) and pre-S1 peptide (PreS/2-48Myr or myrcludex B analog) that suppress the entry of HBV into hepatocytes by targeting NTCP. For the inhibitor screening assay, NTCP-overexpressing HepG2 or Huh-7 cells are treated with either a combination of TCA and an inhibitor (CsA or PreS/2-48Myr) or d4-TCA alone to serve as a reference. The activity of an HBV inhibitor is determined by relative quantification between TCA and d4-TCA in a 1:1 mixture of inhibitor-treated cells and untreated control cells using liquid chromatography-mass spectrometry. With our new approach, the half maximal inhibitory concentration (IC50) values for CsA and PreS/2-48Myr have been determined at micromolar and nanomolar concentrations, respectively, which is consistent with the previous results obtained with other conventional HBV entry inhibitor assay methods. Our assay method does not require HBV infection or radioactive 3H-TCA and provides a facile way to identify viral entry inhibitors via measuring bile acid transport activity of NTCP.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Espectrometria de Massas/métodos , Internalização do Vírus/efeitos dos fármacos , Linhagem Celular Tumoral , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Reprodutibilidade dos Testes , Simportadores/metabolismoRESUMO
Novel analytical platforms for high-throughput determination of lipid turnover in vivo have been developed based on partial metabolic 2H2O labeling. The performance on lipid kinetics measurement of our methods was validated in three different liquid chromatography-mass spectrometry (LC-MS) setups: MS-only, untargeted MS/MS, and targeted MS/MS. The MS-only scheme consisted of multiple LC-MS runs for quantification of lipid mass isotopomers and an extra LC-MS/MS run for lipid identification. The untargeted MS/MS format utilized multiple data-dependent LC-MS/MS runs for both quantification of lipid mass isotopomers and lipid identification. An in-house software was also developed to streamline the data processing from peak area quantification of mass isotopomers to exponential curve fitting for extracting the turnover rate constant. With HeLa cells cultured in 5% 2H2O media for 48 h, we could deduce the species-level turnover rates of 108 and 94 lipids in the MS-only and untargeted MS/MS schemes, respectively, which covers 13 different subclasses and spans 3 orders of magnitude. Furthermore, the targeted MS/MS setup, which performs scheduled LC-MS/MS experiments for some targeted lipids, enabled differential measurement between the turnover rates of the head and tail groups of lipid. The reproducibility of our lipid kinetics measurement was also demonstrated with lipids that commonly detected in both positive and negative ion modes or in two different adduct forms.
Assuntos
Óxido de Deutério/análise , Metabolismo dos Lipídeos , Lipídeos/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Óxido de Deutério/metabolismo , Células HeLa , Humanos , Cinética , Reprodutibilidade dos TestesRESUMO
The energy flow during natural photosynthesis is controlled by maintaining the spatial arrangement of pigments, employing helices as scaffolds. In this study, we have developed porphyrin-peptoid (pigment-helix) conjugates (PPCs) that can modulate the donor-acceptor energy transfer efficiency with exceptional precision by controlling the relative distance and orientation of the two pigments. Five donor-acceptor molecular dyads were constructed using zinc porphyrin and free base porphyrin (Zn(i + 2)-Zn(i + 6)), and highly efficient energy transfer was demonstrated with estimated efficiencies ranging from 92% to 96% measured by static fluorescence emission in CH2Cl2 and from 96.3% to 97.6% using femtosecond transient absorption measurements in toluene, depending on the relative spatial arrangement of the donor-acceptor pairs. Our results suggest that the remarkable precision and tunability exhibited by nature can be achieved by mimicking the design principles of natural photosynthetic proteins.
Assuntos
Transferência de Energia , Peptoides/química , Biomimética , Metaloporfirinas/química , Cloreto de Metileno , Estrutura Molecular , Fotossíntese , Porfirinas/química , Tolueno , Raios UltravioletaRESUMO
The complex chemistry occurring at the interface between liquid and vapor phases contributes significantly to the dynamics and evolution of numerous chemical systems of interest, ranging from damage to the human lung surfactant layer to the aging of atmospheric aerosols. This work presents two methodologies to eject droplets from a liquid water surface and analyze them via mass spectrometry. In bursting bubble ionization (BBI), droplet ejection is achieved via the formation of a jet following bubble rupture at the surface of a liquid to yield 250 µm diameter droplets (10 nL volume). In interfacial sampling by an acoustic transducer (ISAT), droplets are produced by focusing pulsed piezoelectric transducer-generated acoustic waves at the surface of a liquid, resulting in the ejection of droplets of 100 µm in diameter (500 pL volume). In both experimental methodologies, ejected droplets are aspirated into the inlet of the mass spectrometer, resulting in the facile formation of gas-phase ions. We demonstrate the ability of this technique to readily generate spectra of surface-active analytes, and we compare the spectra to those obtained by electrospray ionization. Charge measurements indicate that the ejected droplets are near-neutral (<0.1% of the Rayleigh limit), suggesting that gas-phase ion generation occurs in the heated transfer capillary of the instrument in a mechanism similar to thermospray or sonic spray ionization. Finally, we present the oxidation of oleic acid by ozone as an initial demonstration of the ability of ISAT-MS to monitor heterogeneous chemistry occurring at a planar water/air interface.