ISOLATION AND PURIFICATION OF LEPTOSPIRAL EXTRACELLULAR VESICLES

Abstract

Several pathogenic gram-negative bacteria including Leptospira produce extracellular vesicles (ECVs) possibly derived from bacterial outer membrane.  ECVs harbor multiple active molecules that serve many functions such as secretion system, communication tool, and vaccine candidates. The previous report demonstrated that the chemically induced leptospiral ECV (iECV)-based vaccines conferred protective immunity against lethal infection in the animal model. Therefore, leptospiral ECVs may contain potential vaccine antigens. However, the natural leptospiral ECV (nECV) has not been characterized.  In this study, we isolated and purified both nECV and iECV from Leptospira interrogans serovar Pomona by filtration and ultracentrifugation and then purified by density sucrose gradient. Our results indicated that the nECV and iECV were mainly separated into 50% sucrose fraction. Moreover, the immunoblotting analysis revealed that LipL32, known leptospiral outer membrane lipoprotein, was present in both nECV and iECV. This study should lead to further investigation in the role of ECVs in pathogenesis and identification of new vaccine candidates for leptospirosis.