Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued ...until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.
Mass Spectrometry-Based Metabolomics Kim, Young-Mo; Heyman, Heino M
Methods in molecular biology (Clifton, N.J.),
2018, Letnik:
1775
Journal Article
Metabolomics based on mass spectrometry can provide quantitative and qualitative information of the pool of metabolites (metabolome) present intracellularly or extracellularly in a given biological ...system. A typical metabolomics workflow requires several key steps such as quick and robust sample preparations with quenching of metabolism, chemical derivatization if needed, instrumental measurement, data-processing with/without database information and further statistical analysis and interpretation. Here, we introduce general metabolomics workflows for global and targeted analyses using gas chromatography or liquid chromatography coupled with mass spectrometers.
The utility of metabolomics is well documented; however, its full scientific promise has not yet been realized due to multiple technical challenges. These grand challenges include accurate chemical ...identification of all observable metabolites and the limiting depth-of-coverage of current metabolomics methods. Here, we report a combinatorial solution to aid in both grand challenges using UHPLC-trapped ion mobility spectrometry coupled to tandem mass spectrometry (UHPLC-TIMS-TOF-MS). TIMS offers additional depth-of-coverage through increased peak capacities realized with the multi-dimensional UHPLC-TIMS separations. Metabolite identification confidence is simultaneously enhanced by incorporating orthogonal collision cross section (CCS) data matching. To facilitate metabolite identifications, we created a CCS library of 146 plant natural products. This library was generated using TIMS with N
drift gas to record the
CCS
of plant natural products with a high degree of reproducibility; i.e., average RSD = 0.10%. The robustness of
CCS
data matching was tested using authentic standards spiked into complex plant extracts, and the precision of CCS measurements were determined to be independent of matrix affects. The utility of the UHPLC-TIMS-TOF-MS/MS in metabolomics was then demonstrated using extracts from the model legume
and metabolites were confidently identified based on retention time, accurate mass, molecular formula, and CCS.
Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system ...that mediates the release of macromolecule‐degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non‐conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.
Imaging mass spectrometry (MSI) has contributed important information to several scientific fields and more recently to the biological sciences. The use of MSI as an investigative tool for plant ...metabolomics has recently been explored and initial results show a promising direction that plant metabolomics could be heading to with the use of these mass directed imaging techniques. Matrix assisted laser desorption ionisation (MALDI), secondary ion mass spectrometry and desorption electrospray ionisation mass spectrometry are the best-known MSI techniques used currently. MALDI-MSI is the most common used technique for molecular imaging. With new developments, spatial resolution capabilities have reached 5 μm which has resulted in a significant improvement in the data outputs for MSI analysis. Developments in the improvement of sample preparation techniques, instrumental improvements and more efficient and effective data analysis, have had a dramatic improvement in the obtained informational content. More recent developments on instrumentation and applications are seeing a change in direction towards the imaging of lower mass range metabolites. The unique feature that MSI brings to bio-analytical analysis is the possibility to analyse the distribution of hundreds of plant metabolites across a sample without any significant sample preparation. This has highlighted the potential of MSI techniques as ideal for metabolome analysis with the additional advantage of generating crucial spatial distribution information as well. The further development of MSI for the use in plant metabolomics is at an exciting time and the prospects for interesting new discoveries are very evident.
In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion ...cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO
, CH
, and CO
:CH
porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO
and CH
dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate-peatland feedbacks.
The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their ...mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m/z, isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.
In the wake of the birth of personalized medical treatment, the need for validated in‐home bio‐specimen collection options from patients are required. New ways of collecting and storing bio‐specimens ...to retain important biological information are on the rise. For this reason, dried blood spots (DBSs) have gained increasing attention especially in the field of metabolomics. Metabolomics systematically captures the phenotype of disease, interventions, microbiome, etc. if the most relevant biological pathways are represented broadly, as has traditionally been demonstrated in clinical EDTA plasma. Here we demonstrated results from a study comparing DBS against the golden standard in blood collection, EDTA plasma. The study showed that even though DBS had a reduction of overall metabolites coverage compared to plasma (̴ 800 vs ̴1200 metabolites, respectively), all major pathways and more than 95% of the metabolic sub‐pathways that are routinely detected in plasma are conserved in DBS. Traditional biological signatures, including exercise biomarkers, dietary biomarkers, and even the cyclical trend of the female hormonal biomarkers, are equally well represented in DBS compared to plasma. This evidently showed that metabolic signatures of disease that have been well characterized in plasma are maintained in DBS. The analysis of DBS for metabolomics has come a long way and this study showed DBS to be a valuable alternative to EDTA plasma and ought be considered when plasma is impractical to collect and store i.e., at‐home collections, remote localities, resource limited settings, pediatrics, and IEM screening to name a few. DBS‐based metabolomics provides a powerful tool for personalized medicine and an effective way of bringing metabolomics into our homes.
Due to the recently uncovered health benefits and anti-HIV activities of dicaffeoylquinic acids (diCQAs), understanding their structures and functions is of great interest for drug discovery efforts. ...DiCQAs are analytically challenging to identify and quantify since they commonly exist as a diverse mixture of positional and geometric (cis/trans) isomers. In this work, we utilized ion mobility spectrometry coupled with mass spectrometry to separate the various isomers before and after UV irradiation. The experimental collision cross sections were then compared with theoretical structures to differentiate and identify the diCQA isomers. Our analyses found that naturally the diCQAs existed predominantly as trans/trans isomers, but after 3 h of UV irradiation, cis/cis, cis/trans, trans/cis, and trans/trans isomers were all present in the mixture. This is the first report of successful differentiation of cis/trans diCQA isomers individually, which shows the great promise of IMS coupled with theoretical calculations for determining the structure and activity relationships of different isomers in drug discovery studies.
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