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Paper-based Devices for Isolation and Characterization of Extracellular Vesicles
Published on: April 3, 2015
Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites
Abstract:
At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. We describe here the isolation of these sites from cell membrane fractions of Salmonella anatum. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate and beta-NADH-oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), we infected S. anatum with phage epsilon 15, which causes a rapid change (conversion) in the cell's O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage epsilon 34. We found that the fractions of intermediate density (Int. M) from briefly converted cells bound both phage epsilon 34 and E2-specific antibody. In the electron microscope, epsilon 34 was seen to have absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. Furthermore, the Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. In addition, Int. M material exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. Our data indicate that this membrane fractionation allows one to isolate from Int. M regions a variety of activities associated with adhesion sites.
Insights
Researchers isolated bacterial adhesion sites between the outer and inner membranes (OM and IM) of gram-negative bacteria. These sites, crucial for membrane insertion and phage infection, were identified using bacteriophage epsilon 15 in Salmonella anatum.
Area of Science:
- Microbiology
- Bacterial Cell Biology
- Membrane Biology
Background:
- Adhesion sites between the outer membrane (OM) and inner membrane (IM) are critical for gram-negative bacteria.
- These sites are involved in the insertion of newly synthesized membrane components and bacteriophage infection.
Purpose of the Study:
- To isolate and characterize the adhesion sites between the OM and IM in Salmonella anatum.
Main Methods:
- Isolation of membrane vesicles from Salmonella anatum using sucrose density gradients.
- Infection with bacteriophage epsilon 15 to induce O-antigen conversion and mark lipopolysaccharide synthesis sites.
- Characterization of isolated membrane fractions using enzymatic assays (2-keto-3-deoxyoctonate, beta-NADH-oxidase, phospholipase A1/A2) and bacteriophage binding assays.
- Electron microscopy to visualize bacteriophage absorption to membrane fractions.
Main Results:
- Membrane fractions of intermediate density (Int. M) from briefly converted cells showed specific binding to bacteriophage epsilon 34 and E2-specific antibodies.
- Electron microscopy confirmed significant absorption of bacteriophage epsilon 34 to the Int. M fraction of converted cells.
- Int. M fractions exhibited elevated phospholipase A1 and A2 activities, similar to the OM fraction, while the IM fraction had minimal phospholipase activity.
Conclusions:
- The study successfully isolated bacterial adhesion sites between the OM and IM.
- These isolated sites possess specific molecular markers and enzymatic activities relevant to their function.
- The findings provide a method for studying the molecular mechanisms of membrane assembly and bacteriophage interactions at these critical bacterial sites.
