Dataset Information

Cells responding to bacterial pore-forming toxins release extracellular vesicles displaying similar proteomic content

ABSTRACT: The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) was proposed as a mechanism to eliminate PFTs pores and restore PM integrity. We show here that indeed PFTs are at least partially eliminated through EV release and hypothesized that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different PFTs, Listeriolysin O and Pneumolysin, and determined their proteomic repertoire by LC-MS/MS analysis. Intoxicated cells release similar EVs irrespectively of the PFT used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 76 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further studies. We reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Rupert Mayer

LAB HEAD: Sandra Sousa

PROVIDER: PXD037166 | Pride | 2023-02-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 40

Publications

Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins.

Alves Sara S Pereira Joana M JM Mayer Rupert L RL Gonçalves Alexandre D A ADA Impens Francis F Cabanes Didier D Sousa Sandra S

Toxins 20221220 1

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to e ...[more]

PMID: 36668824

Similar Datasets

Project description:Extracellular vesicles (EVs) are small membrane-derived vesicles that shuttle proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the CNS. Exosomes are small EVs (40-150 nm) that are derived from multi-vesicular bodies (MVBs) of the endo-lysosomal pathway, formed by inward budding of the limiting membrane into the MVB lumen and released into the extracellular space upon fusion of the MVB with the plasma membrane (PM). Previous research revealed certain effector molecules to be required for exosome release. For instance, RAB GTPases have been shown to control exosome release in a cell- and tissue-specific manner. In addition, exosome release appears to be evoked by membrane depolarization in conjunction with calcium influx and to depend on members of the SNARE family of proteins. However, the specific cellular and molecular factors that regulate neuronal exosome release and segregate it from the release of neurotransmitter vesicles are currently unknown. Here, we used a combination of molecular biology, patch-clamp electrophysiology and pH-sensitive dye imaging to examine the effect of the neuronal growth and differentiation factors basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on neuronal EV release. We found that bFGF-treatment of cultured hippocampal neurons increased the abundance of EVs in the culture medium as measured by Western blot and nanoparticle tracking analysis (NTA), without affecting the number or size of neuronal MVBs. The effect of bFGF depended on calcium and on receptor tyrosine kinase (RTK) activity as the calcium chelating agent BAPTA and the tyrosine kinase inhibitor genistein both abolished the bFGF-induced increase in EV release. In accord with these results, untreated neurons had a low rate of spontaneous and stimulus-evoked MVB-PM fusion events as measured by pH-sensitive dye imaging in conjunction with patch-clamp electrophysiology, but treatment with bFGF significantly increased the number of neurons exhibiting MVB-PM fusion events in response to a high-frequency stimulus. Proteomic analysis of neuronal EVs by Liquid Chromatography Mass Spectrometry (LC-MS) demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs, whereas VAMP2 was decreased. Conversely, knocking-down VAMP3 in cultured neurons abolished the effect of bFGF on EV release. Similar to bFGF, the classical neurotrophins BDNF and NGF increased neuronal EV release in a VAMP3-dependent manner. In summary, our results thus reveal a new function for neurotrophic factor signalling in controlling neuronal exosome release and support the investigation of growth factor-mediated signal transduction via EVs in the healthy and diseased CNS.

			Action	DRS
	EVS_PLY_2.raw	Raw
	EVs_1_Joana_Pereira_TR1.raw	Raw
	EVs_1_Joana_Pereira_TR2.raw	Raw
	EVs_1_Joana_Pereira_TR3.raw	Raw
	EVs_1_Sara_Alves_TR1.raw	Raw