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Proteomic analysis for microvesicles isolated from uninfected or M.ab-infected RAW 264.7 cells

ABSTRACT: Microvesicles (MVs) are a subtype of extracellular vesicle (EVs) released by cells, which play important roles in cell-cell communication. The role of host cell-derived MVs in host-pathogen interactions remains to be defined in the context of mycobacterial infection. In this study, we characterized MVs that were isolated from mouse RAW 264.7 cells either uninfected or infected with Mycobacterium abscessus (M.ab), one of the most common non-tuberculous mycobacterial (NTM) pathogens in humans, in cell culture. The proteomic profile of purified MVs was determined by mass spectrometry (MS)-based proteomics.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Carlyn Guthrie

LAB HEAD: Yong Cheng

PROVIDER: PXD062573 | Pride | 2025-11-17

REPOSITORIES: Pride

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			Action	DRS
	1999_asTR_proteinGroups.txt	Txt
	Mab_infected_1.raw	Raw
	Mab_infected_2.raw	Raw
	Mab_infected_3.raw	Raw
	Uninfected_1.raw	Raw

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Publications

Host Cell-Derived Extracellular Vesicles Regulate Iron Uptake in Recipient Macrophages during Mycobacterium abscessus Infection.

Ramos-Wolfley Aidaly Daniela AD Speelmon Charlie A CA Zhang Jing J Guthrie Carlyn M CM Clark Olivia L OL Hartson Steven D SD Liu Lin L Tan Xuejuan X Cheng Yong Y

Journal of proteome research 20251022 11

The role of host cell-derived extracellular vesicles (EVs) in host-pathogen interactions remains to be defined during mycobacterial infection. In this study, we characterized EVs from mouse RAW 264.7 cells uninfected or infected with Mycobacterium abscessus (M.ab), one of the most common nontuberculous mycobacterial (NTM) pathogens in humans. Our results show that M.ab infection increased the release of EVs but had no effect on the size distribution and total p ...[more]

PMID: 41124677

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Mycobacterium abscessus Extracellular Vesicles Increase Mycobacterial Resistance to Clarithromycin in vitro

Project description:Non-tuberculous Mycobacteria (NTM) are a group of emerging bacterial pathogens that have been identified in cystic fibrosis (CF) patients with microbial lung infections. The treatment of NTM infection in CF patients is challenging due to the natural resistance of NTM species to many antibiotics. Mycobacterium abscessus (M. abscessus) is one of the most common NTM strains found in the airway of CF patients. In our current study, we characterized the extracellular vesicles (EVs) released by drug-sensitive M. abscessus untreated or treated with clarithromycin, one of the well-known anti-NTM drugs. Our data show that clarithromycin treatment increases mycobacterial protein trafficking into EVs as well as the secretion of EVs in M. abscessus culture. Additionally, EVs released by clarithromycin-treated M. abscessus increase M. abscessus resistance to clarithromycin when compared to EVs from untreated M. abscessus. EV proteomic analysis further indicates that EVs released by clarithromycin-treated M. abscessus carry an increased level of 50S ribosomal subunits, the target of clarithromycin. Taken together, our results suggest that mycobacterial EVs play an important role in increasing M. abscessus resistance to clarithromycin treatment.

2025-05-06 | PXD047862 | Pride

Expression Profiling of Extracellular Microvesicles Reveals Intercellular Transmission of Oncogenic Pathways

Project description:Circulating microvesicles (MVs) have been described as important players in cell-to-cell communication carrying biological information both in normal and pathologic condition. MVs released by cancer cells may incorporate biomolecules such as active lipids, proteins and RNA, which can be delivered and internalized by recipient cells potentially altering gene expression of receiving cells eventually impacting disease progression. In this study, we took advantage of a leukemia in vitro model to investigate MVs as vehicles of protein coding messages. Leukemic cell lines (K562, REH and SHI-1) carrying recurrent translocations were analyzed. In the leukemic cells these translocations are transcribed into oncogenic fusion transcripts. Here, using gene expression microarrays we monitored leukemic fusion transcripts as hallmarks of leukemic cells transcriptome to track mRNA transfer from parental cells to MVs. Transcriptome analysis of K562 cells and released MVs disclosed MVs as not just “small scale cells”. In fact, a number of transcripts related to membrane activity, cell surface receptors and extracellular communication were enriched in the MVs pool. On the other hand, sets of transcripts related to the basal cellular functions and transcripts of the BCR-ABL oncogenic pathway downstream of the fusion protein were detected in MVs as well as in parental K562 cells. Moreover, through co-culture analyses uptake of leukemic MVs in receiving cells was confirmed and an MV-dosage dependent increase of target cell proliferation was demonstrated. Our study provides novel understanding of tumor-derived MVs as carrier of protein coding messages. For the first time a whole transcriptome gene expression analysis has been performed comparing K562 parental cells and released MVs, showing an enrichment of transcripts coding for oncogenic proteins and for proteins involved in key cellular pathways.

2017-04-14 | GSE78718 | GEO

Synthesis and Anti-mycobacterial Activity of Novel Medium-chain β-Lactone Derivatives: A Multi-Target Strategy to combat Mycobacterium abscessus and other Pathogenic Mycobacteria

Project description:Targeting the enzymes involved in mycobacterial lipid metabolism; which are mainly serine and cysteine enzymes (i.e., (Ser/Cys)-based enzymes; has emerged as a promising strategy to combat not only M. tuberculosis, but also other chronic non-tuberculous mycobacterial infections like those caused by M. marinum or M. abscessus. Among the known inhibitors of such mycobacterial enzymes, β-lactones represent promising candidates for the development of new derivatives that target lipid-processing enzymes critical for mycobacterial growth and survival. In this context, a new set of 38 lipophilic β lactone derivatives have been synthesized and tested for their anti-mycobacterial activities against the three aforementioned pathogenic mycobacteria. Remarkably, the determined MIC revealed that some VM β-lactones were able to inhibit M. abscessus growth in vitro in culture broth medium and/or inside infected macrophages. In addition, using a competitive activity-based protein profiling approach, the potential target enzymes of VM043, the most active inhibitor of extracellular bacterial growth, were further identified; thus, confirming the multi-target nature of this family of molecules.

2025-04-23 | PXD057836 | Pride

TLR4 senses oxidative stress mediated by partially oxidized microvesicles.

Project description:Oxidative stress is a hallmark of inflammation in infection or sterile tissue injury. We show that partially oxidized phospholipids of microvesicles (MVs) from plasma of patients with rheumatoid arthritis or cells exposed to oxidative stress induce activation of TLR4. MVs from healthy donors or reconstituted synthetic MVs can be converted to TLR4 agonists by limited oxidation, while prolonged oxidation abrogates the activity. Activation by MVs mimics the mechanism of TLR4 activation by LPS. However, LPS and MVs induce significantly different transcriptional response profile in mouse BMDMs with a strong inflammation-resolving component induced by the endogenous signals. MVs thus represent a ubiquitous endogenous danger signal released under the oxidative stress, which underlies the pervasive role of TLR4 signaling in inflammation.

2015-06-01 | GSE45234 | GEO

Single cell spatial transcriptome and RNA-seq analysis in human lung tissues with mycobacterial infection: Single cell spatial transcriptome

Project description:Tuberculosis and non-tuberculous mycobacterial (NTM) diseases are infections caused by Mycobacterium tuberculosis and non-tuberculous mycobacteria, leading to the formation of granulomatous lesions with caseous necrosis in the lungs. We applied spatial transcriptomics at single-cell resolution (CosMx Spatial Molecular Imaging) to human lung samples from patients with mycobacterial infections. RNA-seq was also performed on the samples.

2025-08-29 | GSE276083 | GEO

Single cell spatial transcriptome and RNA-seq analysis in human lung tissues with mycobacterial infection: RNA-seq analysis

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Microvesicles derived from human mesenchymal stem cells protect against acute tubular injury

Project description:Administration of exogenous mesenchymal stem cells (MSCs) has been shown to improve the recovery from acute kidney injury (AKI). It has been suggested that the beneficial effect of MSCs is related to the paracrine release of factors favouring proliferation of intrinsic epithelial cells survived to injury rather than to their trans-differentiation. However the factors involved remain to be determined. In the present study we demonstrated that microvesicles (MVs) derived from human bone marrow MSCs are able to stimulate in vitro proliferation and apoptosis resistance of tubular epithelial cells (TEC). In addition, MVs were found to accelerate in vivo the morphological and functional recovery of glycerol induced AKI in SCID mice by inducing TEC proliferation. The effect of MVs on the recovery of AKI was comparable to that of human MSC treatment. In vitro we found that the CD44 and beta1-integrin-dependent incorporation of MVs in TEC was required for their biological action. However, despite their internalization, RNase-treated MVs failed to induce in vitro apoptosis resistance and TEC proliferation, and in vivo recovery from AKI, suggesting an RNA-dependent biological effect. Microarray analysis and quantitative RT-PCR of MV-RNA extract indicated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated with the mesenchymal differentiative phenotype and with several cell functions involved in the control of transcription, proliferation, apoptosis and cell immune regulation. These results suggest that MVs derived from MSCs may activate a proliferative program in TEC survived to injury in AKI by an horizontal transfer of mRNA.

2009-01-20 | GSE12243 | GEO

Trancriptional profiling in microvesicles in stimulated (M1 and M2) and unstimulated conditions

Project description:Extracellular membrane vesicles (MVs) are powerful biomarkers in several pathological processes. The potential advantage of MVs relays on the assumption that their content reflects processes ongoing in pathologically relevant cell types. Using microarrays, we performed transcriptional profiling in stimulated (M1 and M2) and unstimulated conditions.

2016-06-03 | E-MTAB-1540 | biostudies-arrayexpress

Bone marrow mesenchymal cells from Myelodisplastic Syndromes release microvesicles that incorporate into CD34+ cells and may modify their behaviour.

Project description:The secretion of exosomes/ microvesicles (MVs) is a recently described mechanism of intercellular communication that is based on the transfer of bioactive molecules. Our hypothesis was that mesenchymal stromal cells (MSC) from myelodisplastic syndromes (MDS) patients could modify CD34+ hematopoietic progenitor cells (HPC) properties throughout MVs. The latter were isolated from MSC from MDS patients and healthy donors (HD). MVs were purified by ExoQuick-TC exosome precipitation solution or ultracentrifugation and identified by transmission electron microscopy (TEM) and flow cytometry (FC). Their Micro-RNA content was evaluated by microarrays. Incorporation of MVs into CD34+ cells was analyzed by FC and confocal microscopy (CM). Our results showed that MVs displayed the same immunophenotypic pattern and similar morphology. The content of miRNAS in the MVs from MDS and HD was significantly different. Next, MVs incorporation into HPC was observed, being highest after 24 hours of incubation. We demonstrated that MVs could modify the HPC behavior since the incubation of HPC with MVs led to gene expression modification evaluated by RT-PCR and increased cell viability by FC. In summary, we show that BM-MSC produce MVs with different cargo in MDS compared to HD. These structures can incorporate into HPC and modify their properties.

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Characterization of Exosomes Released from Mycobacterium abscessus-infected Macrophages

Project description:Extracellular vesicles, such as exosomes, play a critical role in cell-to-cell communication and have been found to modulate cellular processes, including metabolic and inflammatory pathways, in recipient cells. Non-tuberculous Mycobacteria (NTM), such as Mycobacterium abscessus (M.ab), are a group of environmental bacteria that can cause severe lung infections in populations with pre-existing lung conditions, such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). There is limited knowledge of the engagement of extracellular vesicles in the host-pathogen interactions in the context of NTM infections. In this study, we found that M.ab infection increased the release of a subpopulation of exosomes (CD9, CD63 and/or CD81 positive) by mouse macrophages but did not affect exosome morphology. Proteomic analysis of the vesicles demonstrated that M.ab infection affects the enrichment of host proteins in exosomes released by macrophages. When compared to exosomes from uninfected macrophages, exosomes released by M.ab-infected macrophages significantly improved M.ab growth and downregulated the intracellular level of glutamine in recipient macrophages in cell culture. Interestingly, the protein abundance of the glutamine transporters Slc1a5 and Slc38a2 were found to be enriched in exosomes from M.ab-infected macrophages compared to those from uninfected macrophages. Increasing glutamine concentration in the medium rescued intracellular glutamine levels and inhibited M.ab growth in recipient macrophages treated with exosomes from M.ab-infected macrophages. Taken together, our results indicate that exosomes may serve as extracellular glutamine eliminators that interfere with glutamine-dependent M.ab killing in recipient macrophages. This suggests that the development of exosome-targeting therapies may have the potential to augment current treatments for mycobacterial infections.

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