Abstract
Recently, membrane vesicle (MV) production was described in Gram-positive bacteria, which harbor a variety of components such as toxins, antibiotic resistance proteins, proteases, DNA, and immune modulators. Free lipids have the ability to form micelles, thus it is important to rule out spontaneous association of lipids into vesicle-like structures and rather, that MVs are produced naturally by a metabolically active cell. Here, we describe a protocol utilizing the polysaccharide, glucuronoxylomannan (GXM) from Cryptococcus neoformans (C. neoformans) as a marker to differentiate naturally produced MVs from vesicles that form spontaneously in the Gram-positive model organism, Bacillus subtilis (B. subtilis). MVs are purified from bacterial cultures grown in the presence of GXM; MVs naturally produced by cells would not contain GXM in the lumen whereas vesicular structures forming in the media could encapsulate GXM and this can be visualized via immunogold transmission electron microscopy.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Amicon ultrafiltration setup. The ultrafiltration cells are connected to compressed nitrogen gas. The gas applies pressure, forcing the supernatant through the 100 kDa filter membrane. Vesicles stay in the concentrated supernatant while smaller proteins and liquid supernatant flow into waste container. Figure 2. Beckman ultracentrifuge tubes. Ultracentrifugation tubes utilized to pellet vesicles. Figure 3. Transmission electron micrograph of immunogold labeling of GXM in vesicles from Bacillus stubtilis. Micrograph shows vesicles and gold particles. Gold particles indicate the presence of GXM. Notice all of the gold is outside of vesicles. White arrows indicate vesicles and black arrows indicate gold particles. Scale bar=100 nm Vesicles are not incredibly stable so it is suggested to process them immediately after isolation. Only Beckman polyallomer/propylene ultracentrifugation tubes can be used for IEM preparation.Polycarbonate tubes cannot be used for this procedure but can be used when purifying vesicles for other experiments. The sonicated (unnatural) vesicles serve as a positive control. It is expected that vesicles from the sonicated vesicle preparation will contain significantly more gold particles than the naturally produced vesicles. One may also expect that sonicated vesicles have a smaller and less variable diameter than that of natural vesicles.
Notes
B. subtilis strain 168 concentrates very quickly but other strains of bacteria may not. B. subtilis strain 168 produces a large quantity of recoverable vesicles and only requires 100 ml of culture whereas other bacteria may require volumes larger than 1 L. Concentrating to small volumes allows for less ultracentrifugation spins and cuts down on the use of the ultracentrifuge tubes. Ultracentrifuge tubes can be washed and re-used but be aware there may be some contamination.
Recipes
Acknowledgments
Funding from NIH Grant Numbers: HL059842, AI033774, AI033142, AI052733 and Center for AIDS Research at Albert Einstein College of Medicine. Protocol is adapted from Brown et al. (2014) and vesicle purification is adapted from Prados-Rosales et al. (2014).
References
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