Production of Outer Membrane Vesicles From Acetic Acid Bacteria and Their Properties

Abstract

Outer membrane vesicles (OMVs) are mainly composed of lipopolysaccharides (LPS), phospholipids, outer membranes, and periplasmic proteins. Recently OMV vaccines have been developed in which LPS acts as an adjuvant. However, attenuation of the toxicity of typical LPS is necessary to reduce the adverse effects of the OMV vaccine. We have previously found that the acetic acid bacterium Acetobacter pasteurianus produces LPS with low immunostimulatory activity. In this study, we isolated OMVs from Acetobacter pasteurianus and analyzed its immunostimulatory activity. A. pasteurianus NBRC 3283 was cultured at 27°C in 804 broth and vesicle secretion from the cells was observed by TEM imaging after 2 days of culture. Vesicles were separated from the culture medium after 7 days of culture by ultracentrifugation. The results showed that the precipitate contained vesicles that could be purified by OptiPrep density gradient centrifugation. As the vesicles were composed of LPS and outer membrane proteins, we determined that they were OMVs and named them Ap-OMVs. Furthermore, we found that Ap-OMV stimulated toll-like receptor 2 (TLR2) and weakly stimulated TLR4 in TLR-expressing cells and J774A.1 cells. Furthermore, OMV-like vesicles were detected in Japanese black vinegar. These data suggest that A. pasteurianus produces OMVs containing LPS and stimulates the immune system.