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.
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.
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 manner2,3,4. 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 the culture filtrates of mycobacteria grown in an iron-rich medium1,5,6,7,8.
However, previous work demonstrated that iron limitation greatly stimulates vesicle release in Mtb,possibly to capture iron via mycobactin, a siderophore secreted in MEVs9. Although procedures for MEVs isolation from Mtb cultured in high iron medium have been described, an efficient methodology to obtain MEVs from low iron cultures has not been reported. Therefore, the goal of this method is to isolate, purify, and quantify MEVs obtained from low iron cultures so that they can be used for biochemical and functional assays and for the analysis of genetic determinants of vesicle production in mycobacteria.
1. Preparation of Iron-depleted Defined Medium
2. Growing Mycobacteria in Iron-limited Conditions
3. Collection of MEVs
4. Isolation of MEVs
5. Quantification of MEVs
6. Qualitative analysis of MEVs
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 (Figure 2, Figure 3). Protein and lipid concentration normalized to colony-forming units (CFUs) showed an approximately eightfold increase of MEV yield in low iron relative to high iron conditions (50 µM FeCl3) (Figure 3). Although the results of one representative experiment is presented, this is a highly reproducible result based on multiple (>10) isolations of MEVs. The pure MEV yield obtained from a 1 L low iron culture by this method was approximately 500 µg of protein.
Figure 1: Diagrammatic representation of the methodology used for MEV purification and quantification. Mycobacteria grown in agar plates were used to inoculate iron-depleted minimal medium and grow Mtb for EV isolation. MEVs were purified by a discontinuous density gradient from the cell-free culture filtrate. A combination of membrane lipid and vesicle protein determination, microscopy, and nanoparticle analysis was implemented to characterize purified MEVs. Please click here to view a larger version of this figure.
Figure 2: Characterization of purified MEVs. (A) Shown are photographs of an actual density gradient separation of crude MEVs and the pellet of purified MEVs collected by ultracentrifugation of gradient fraction 3 (F3). (B) SDS-gel stained showing the protein profile of the various density gradient fractions. (C) Dot blot analysis showing vesicle-associated proteins concentrated in F3. (D) MEVs present in F3 observed by negative staining. (E) MEV size distribution according to nanoparticle analysis (NTA). Please click here to view a larger version of this figure.
Figure 3: Comparative analysis of MEV yield in low and high iron cultures. A representative result of (A) protein and lipid quantification and (B) dot blot analysis of purified MEVs isolated from iron-limited and iron sufficient Mtb cultures. Please click here to view a larger version of this figure.
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 isolation methods. Because mycobacteria release glycolipids and other hydrophobic molecules that aggregate and easily contaminate crude MEV preparations into the medium, it is important to purify and validate MEVs before conducting biochemical and functional studies. Based on previous observations that demonstrated that Mtb enhances the release of MEVs under conditions of iron limitation, a protocol was established for EV purification from iron-limited mycobacteria. It has also been confirmed that non-virulent M. smegmatis also increases release of EVs in response to low iron conditions (data not shown). Therefore, the same protocol could be used to purify EVs from this bacterium in BSL-2 conditions.
A critical step of this procedure is the preparation of the low iron medium. This medium should be prepared as described here and stored in a plastic container, not in glass, to prevent iron contamination. Supplements commonly used in Mtb growth medium to stimulate bacterial growth and prevent characteristic mycobacterial clumping, such as bovine serum albumin, Tween-80, or tyloxapol, must be avoided. These additives lead to lipoprotein complex artifacts that copurify with vesicles and reduce vesicle yield. For CFU determination, a small culture in medium supplemented with detergent (Tween-80 or tyloxapol) can be set in parallel to the detergent-free large culture. MEVs in the culture filtrate are stable at 4 °C for several days. Therefore, if not processed immediately, the culture filtrate can be stored refrigerated.
The total yield of purified MEV from 1 L of culture was around 500 µg/L protein, which is sufficient to conduct multiple analyses such as proteomics, lipidomics, and functional assays. Depending on the type of assay, sufficient MEVs can be isolated from smaller volumes (i.e., 250 mL). This facilitates comparative analysis of conditions and factors influencing MEV release.
This is an effective method to purify MEVs, but it has limitations. It is a long procedure with multiple ultracentrifugation steps. In the future, this method will be compared to gel filtration chromatography, and as molecular markers of MEVs are discovered, affinity capture methods could be implemented. The host environment is iron-limited, therefore MEVs produced by Mtb in a low iron medium are probably more closely related to MEVs produced during infection and could provide relevant insights about the role of MEVs in tuberculosis pathogenesis.
The authors have nothing to disclose.
We are grateful to Rafael Prados-Rosales for sharing the anti-MEV antisera and Navneet Dogra for performing nanoparticle tracking analysis.
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 x 89 mm | Beckman Coulter | 355618 | Polycarbonate ultra centrifugation tubes 25 x 89 mm |
Polypropylene thin walled centrifuge tube 13×15 mm | Beckman Coulter | 344059 | Polypropylene thin walled centrifuge tube 13×15 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 |