Methods for Studying Fusion of Bacterial Extracellular Vesicles with Intact Bacteria and Host Cells

Bacterial extracellular vesicles (BEVs) are nano- or micrometer-sized membrane-bound lipid vesicles released from both Gram-negative and Gram-positive bacteria. Cellular transport, communication, pathogenesis, and host-pathogen interactions are some of the major biological processes impacted by BEVs. Among these, host-pathogen interactions and bacterial pathogenesis are emerging as highly important targetable avenues underlined by the issues of antimicrobial resistance, thus demanding novel targets and approaches to treat bacterial infections. In this aspect, the study of the interaction of BEVs with bacteria and/or host cells becomes imperative and brings the membrane fusion process to the forefront. Furthermore, membrane fusion also underscores the performance of BEVs as nano-therapeutic delivery platforms. Here, we report methods to study fusion kinetics between mycobacteria-derived extracellular vesicles, which we refer to as MEVs, and intact mycobacteria or MEVs themselves. We also discuss the isolation of MEVs and their characterization. We outline critical factors that affect fusion kinetics by MEVs. The same principle can be extended for studying fusion between BEVs and mammalian host cells important for understanding how BEVs influence host-pathogen crosstalk.