Published: March 31st, 2021
Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.
The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of "large" (350 nm) OMVs and "small" (<150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.
Gram-negative bacteria release vesicles derived from their outer membrane, so-called outer membrane vesicles (OMVs), throughout growth. These OMVs play important roles in cell-to-cell communication, both between bacteria and host as well as between bacterial cells, by carrying a number of important biomolecules, including DNA/RNA, proteins, lipids, and peptidoglycans1,2. In particular, the role of OMVs in bacterial pathogenesis has been extensively studied due to their enrichment in certain virulence factors and toxins3,4,5....
1. Preparation of buffers
Figure 2 shows representative results from this method. OMVs produced by A. actinomycetemcomitans strain JP2 were first purified from the culture supernatant using ultracentrifugation15. We previously found that this strain produces two populations of OMVs, one with diameters of about 300 nm and one with diameters of about 100 nm15. To separate these OMV populations, we purified the sample using the SEC protocol described above. E.......
Here, we have provided a protocol for the simple, fast, and reproducible separation of bacterial OMV subpopulations. Although the technique is relatively straight-forward, there are some steps that must be performed extremely carefully to ensure that efficient separation occurs in the column. First, it is essential that the gel be loaded into the column carefully and slowly to avoid air bubbles. We have observed that leaving the gel at room temperature for several hours before loading the column allows the gel to equilib.......
|1-Step Ultra TMB-ELISA
|Amicon 50 kDa filters
|Bovine Serum Albumin (BSA)
|ELISA Immuno Plates
|1.5 x 50 cm
|Infinite 200 Pro Plate Reader
|Potassium Chloride (KCl)
|Potassium Phosphate Monobasic Anhydrous (KH2PO4)
|Sephacryl S-1000 Superfine
|Sodium Chloride (NaCl)
|Sodium Phosphate Dibasic Anhydrous (Na2HPO4)
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