Dataset Information

Establishment of Minimum Protein Standards for Mycobacterium tuberculosis-Derived Extracellular Vesicles through Comparison of EV Enrichment Methods

ABSTRACT: Extracellular vesicles (EVs) produced by Mycobacterium tuberculosis, the causative agent of tuberculosis, are involved in acquiring nutrients, evading host immune mechanisms, and modulating the host immune response. The functions of these M. tuberculosis EVs (MEVs)are hypothesized to be due to the dynamic composition of MEVs. However, there are various methods used to purify MEVs, and these methods can impact MEV characteristics such as size, purity, integrity, and composition. In this study, we investigated several methods for MEV purification, including: ultracentrifugation, differential density gradient-based ultracentrifugation, qEV size exclusion chromatography, and Capto(TM)Core size exclusion chromatography. We evaluated the size, concentration, appearance, purity, and protein composition (by mass spectrometry) of MEVs from each of these methods. We ultimately found that protein and particle yield vary across methods and that the size exclusion chromatography-based methods are more technically reproducible than the ultracentrifugation-based methods. Through mass spectrometry analysis, we also identified seven M. tuberculosis proteins that were present regardless of MEV enrichment method, that we propose could be used as qualitative markers of MEVs versus contaminants (in addition to MEV size and appearance criteria), to benefit the reproducibility of ongoing MEV studies.

INSTRUMENT(S):

ORGANISM(S): Mycobacterium Tuberculosis H37rv

SUBMITTER: Kimberly Shelton

LAB HEAD: Karen M Dobos

PROVIDER: PXD056465 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	100FT_sample_1_run1.d.zip	Other
	100FT_sample_1_run1.mzXML	Mzxml
	100FT_sample_1_run1.pepXML	Pepxml
	100FT_sample_1_run2.d.zip	Other
	100FT_sample_1_run2.mzXML	Mzxml

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Publications

Establishment of minimum protein standards for <i>Mycobacterium tuberculosis</i>-derived extracellular vesicles through comparison of EV enrichment methods.

Ryan Joan M JM Shelton Kimberly K Dzieciatkowska Monika M Kruh-Garcia Nicole N Dobos Karen M KM

Mycobacteria (London, England) 20250415 1

<i>Mycobacterium tuberculosis</i> extracellular vesicles (MEV) have been described as having potent immunological activities that are both beneficial and harmful to the host. Key to understanding this conflicting information is the proteomic characterization of MEVs. However, there is neither a standard for a purification method nor markers to assess relative purity and quality of MEVs. In this study, we purified MEVs by four different methods (simple ultracentrifugation, differential density gr ...[more]

PMID: 40256639

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Project description:Tuberculosis (TB) still represents a major global health problem affecting over 10 million people worldwide. At present, there is an urgent need for defining prognostic parameters, differential diagnostic, and correlates of protection from the disease. The gold standard test for TB diagnosis is still smear microscopy, but insufficiently detects pulmonary disease, besides the necessity of having laboratory infrastructures and be time-consuming. Both LAM urine test and Xpert MTB/RIF have been major game changers in this context. However, the low sensitivity of the former and the considerable delay of sample delivery of the latter, make the improvement of the TB diagnostic landscape a priority. The development of rapid point-of-care (POC)-based testing approaches would represent an important breakthrough in TB diagnostics in view of these short-comings. Most forms of life produce extracellular vesicles (EVs) and since the first detection of bacterial EVs more than 60 years, subsequent studies have demonstrated their functional commonality despite differences in bacterial cell envelope architecture. We demonstrated that Mycobacterium tuberculosis (Mtb), the causative agent of TB, produces EVs in vitro and in vivo as part of a sophisticated mechanism to manipulate host cellular physiology and to evade the host immune system. In a previous serology study, Mtb EVs (MEVs) were used to investigate their potential role as biomarkers to discern between different forms of disease status. It was shown that the recognition of several MEV associated proteins could have diagnostic properties. In this study, we pursued to expand the capabilities of MEVs in the context of TB diagnostics by analyzing the composition of MEVs isolated from Mtb cultures submitted to iron starvation and, testing their immunogenicity against a new cohort of serum samples including TB+, LTBI and healthy donors. We found that MEVs differ in protein composition when Mtb is under host-related stress yet MEVs seem to carry antigens that could serve as bonafide markers for direct detection. In addition, TB serology revealed three new MEV antigens with great biomarker capacity. These results encourage investigating the feasibility of the development of a POC device based on selected MEV-associated proteins.

Project description:Milk-derived extracellular vesicles (mEVs) have been proved to play a critical role in intercellular communication, mainly through the microRNAs (miRNAs) that they carry, to regulate biological functions of the target cells. Given miRNAs are evolutionarily conserved, EVs present in commercial milk may play a role in the physiology and health consumers. It is therefore essential to know the effects of technological treatments such as skimming and spray drying on the EV content of milk powders and on the cargo of bioactive molecules, in particular miRNAs, that they convey. Since goat’s milk or goat milk based formulas are considered as a healthy alternative for infants with cow’s milk sensitivities, including allergy, we undertook to analyze the EV content of skimmed and unskimmed goat's milk powders and to characterize their RNA content, in particular their miRNomes. mEVs were isolated using an optimized protocol based on Size Exclusion Chromatography (SEC) and compared regarding morphology, number and size by Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Their RNA and protein content were determined and their miRNomes established, using RNA sequencing. In this study we demonstrated that goat milk powders, skimmed or not upstream the spray drying treatment, contained many mEVs, ranging from 5.4 1011 to 2.5 1012 particles per mL of reconstituted milk, with an average size between 136.8 and 160.6 nm. We also demonstrated that mEVs carried significant amounts of RNA, including miRNAs. Using RT-qPCR, mRNAs encoding five of the major milk proteins were detected, suggesting that mEVs originated from mammary epithelial cells. We established the goat milk powder miRNome by identifying 351 miRNAs of which 233 are common to the 262 miRNAs previously profiled in raw goat milk. The 20 most abundant miRNAs (TOP 20) account for 80% of the total reads and the hierarchy of this TOP 20 miRNAs is somewhat overturned when comparing goat milk powder and raw goat milk. Surprisingly, whereas the comparison of raw from cow and goat milk confirmed the prevalence of miR-148a, miR-21-5p and miR-26a/miR-30a-5p, let-7a-5p and let-7f, which occupied ranks 1 and 2, respectively, in powders, were relegated to ranks 6 and 10 and 5 and 11 in raw goat and cow milk, respectively. Conversely to what was previously reported, we provide evidence that: i) EVs of typical morphology are present in goat milk powders; ii) mEVs survived the technological processes used to produce the powders; iii) their miRNA cargo is protected from degradation even though their miRNomes are not an exact mirror of miRNomes of EVs derived from fluid raw milk.

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Dataset Information

Establishment of Minimum Protein Standards for Mycobacterium tuberculosis-Derived Extracellular Vesicles through Comparison of EV Enrichment Methods

Dataset's files

Publications

Establishment of minimum protein standards for <i>Mycobacterium tuberculosis</i>-derived extracellular vesicles through comparison of EV enrichment methods.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Establishment of Minimum Protein Standards for Mycobacterium tuberculosis-Derived Extracellular Vesicles through Comparison of EV Enrichment Methods

Dataset's files

Publications

Establishment of minimum protein standards for &lt;i&gt;Mycobacterium tuberculosis&lt;/i&gt;-derived extracellular vesicles through comparison of EV enrichment methods.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Establishment of minimum protein standards for <i>Mycobacterium tuberculosis</i>-derived extracellular vesicles through comparison of EV enrichment methods.