Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Summary

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Abstract

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Introduction

Mycobacterial extracellular vesicles (MEVs) are membrane-bound nanoparticles, 60−300 nm in size, naturally released by fast- and slow-growing mycobacteria¹. MEVs released by pathogenic mycobacteria constitute a mechanism to interact with the host via immunologically active proteins, lipids, and glycolipids secreted in a concentrated and protected manner^2,3,4. To characterize MEVs and understand their biogenesis and functions, strict and efficient methods of vesicle purification and validation are crucial. Thus far, MEVs have been isolated from th....

Protocol

1. Preparation of Iron-depleted Defined Medium

1. Prepare 1 L of minimal medium (MM) by dissolving 5 g of KH₂PO₄, 5 g of L-asparagine, 20 mL of glycerol, and 2 g of dextrose in 900 mL of deionized water in a plastic container. Avoid glass to prevent iron contamination. Adjust the pH to 6.8 with 5 N NaOH and the volume to 1 L with water.
2. Add 50 g of metal chelating resin (MCR) and gently agitate using a magnetic stir bar for 24 h at 4 °C. Sterilize and remove the MCR by filtration through a 0.22 µm filter unit with a plastic receiver. To accelerate filtration and prevent filter clogging, let the resin sediment for abou....

Results

MEVs were purified by differential sedimentation in a density gradient (Figure 1, Figure 2). Under the conditions described, MEVs separated mostly in gradient fraction 3 (F3), which corresponds to 25% iodixanol. This conclusion is based on the detection of protein, membrane lipid, microscopic visualization of intact MEVs, nanoparticle size distribution, and positive reactivity with an antivesicle antiserum (F.......

Discussion

Multiple methods to purify eukaryotic cell-derived exosomes have been developed¹². In contrast, there is limited information on effective methods to purify bacteria-derived EVs⁷. Efficient isolation of Mtb-derived EVs needs to consider the intrinsic difficulties in growing this pathogenic mycobacterium. Mtb has a long division time (~24 h) and should be handled in biosafety level three (BSL-3) conditions. Therefore, it is important to optimize the efficiency of MEV isolatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amicon stirred cell Model 108	EMD Milipore	UFSC40001	Cell Ultrafiltration system
BD Polypropilene 225 mL conical tubes	Fisher	05-538-61	Conical centrifuge tubes
Biomax 100 kDa cut-off ultrafiltration membrane	EMD Milipore	PBHK07610	Ultrafiltration membrane
Chelex-100 resin	Bio-Rad	142-2842	Metal chelating resin
Middlebrook 7H10 Agar	BD Difco	262710	Mycobacterial Agar plates
Middlebrook 7H9 Broth	BD Difco	271310	Mycobacterial broth medium
Nitro cellulose blotting membrane	GE Healthcare	10600001	Blotting Membrane
Optiprep	Sigma	D1556	Iodixanol
Polycarbonate ultra centrifugation tubes 25 mm x 89 mm	Beckman Coulter	355618	Polycarbonate ultra centrifugation tubes 25 mm x 89 mm
Polypropylene thin walled centrifuge tube 13 mm x 15 mm	Beckman Coulter	344059	Polypropylene thin walled centrifuge tube 13 mm x 15 mm
Protein Assay dye	BioRad	5000006	Bradford Protein Staining
SYPRO Ruby	Molecular Probes	S12000	Ultrasensitive protein stain
TMA-DPH	Molecular Probes	T204	1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate
Vacuum filtration flasks	CellPro	V50022	Filter Unit