Background

The membranes of all cells are dynamic structures, the biomolecule composition of which is constantly changing as cells respond to environmental stimuli, alter protein and lipid composition, release waste products, take in nutrients, and perform many other cellular processes (Vereb et al., 2003; Watson, 2015). Throughout cellular life cycles, fragments of the outermost membrane are often shed as nanosized particles. In bacteria, these structures are often referred to as membrane vesicles (MVs) or outer membrane vesicles (OMVs), which will be referred to as MVs throughout for simplicity (a representative example of MVs from Lactobacillus casei is shown in Figure 1). As interest in these biological nanoparticles has grown in recent years, researchers have shown that MVs have broad biological activities, including host-microbe interactions, gene transfer, and community regulation (Kulp and Kuehn, 2010; Caruana and Walper, 2020).

Bacterial MVs have shown significant promise for applications such as vaccine development and potential therapeutic applications. Naturally occurring OMVs from Gram-negative bacteria have shown significant promise in the development of vaccines for bacteria such as Neisseria meningitidis and Burkholderia pseudomallei, pathogens that have proven challenging to vaccine and therapeutic development alike (Holst et al., 2009; Nieves et al., 2014). Recently, research groups have also shown that the MVs from some commensal bacteria can also modulate responses from host immune systems and stimulate host neurological systems (Mata Forsberg et al., 2019; Molina-Tijeras et al., 2019; Rodovalho et al., 2020). With growing capabilities in synthetic biology, the potential uses of MVs are steadily increasing as researchers have developed engineering strategies that allow for modification of genetic and cellular pathways to control the composition of nascent MVs. These efforts have led to new biomaterials for therapeutic applications, environmental decontamination, and other purposes (Alves et al., 2018; Qing et al., 2019).

The classification of lactic acid bacteria (LAB) encompasses several genera of bacteria with similar characteristics of acid-tolerance and fermentation capabilities. Many LAB are classified as generally regarded as safe (GRAS) and have been used in the production of food products for centuries. Additionally, several LAB have been recognized as beneficial to their host and are studied for their health benefits as probiotics leading to a large commercial market for probiotic supplements and foods. While live-bacterial cultures are the most commonly used form of probiotics, the potential for engineering or enriching for specific cellular components has led researchers to explore the use of purified MVs for controlled therapeutic applications (Seo et al., 2018; Molina-Tijeras et al., 2019; Dean et al., 2020).

There are numerous protocols for the purification of MVs from eukaryotic cells and Gram-negative bacteria, which have been the primary focus of MV research (Klimentova and Stulik 2015; Alves et al., 2017; Dauros Singorenko et al., 2017). Recently, there has been growing interest in the MVs of gut bacteria and the roles they may play in host and community interactions. LAB are Gram-positive bacteria and therefore have a significantly different membrane and peptidoglycan structure as compared to Gram-negative bacteria. While this may not specifically contribute to biophysical differences between Gram-negative and Gram-positive MVs, it has been shown that the MVs of some LAB species have a bimodal size distribution with an abundant population of smaller MVs in the 10-50 nm size range (Dean et al., 2019). Here, we describe protocols for the isolation and characterization of MVs that have proven successful for numerous LAB species. MVs are isolated from overnight LAB cultures via ultracentrifugation and then analyzed for concentration and relative size distribution using a NanoSight nanoparticle tracking instrument. Dynamic light scattering (DLS) is used as another way to measure size distribution and also to measure zeta potential (surface charge). Then, the protein content of MVs can be examined both qualitatively using SDS-PAGE and specifically by using mass spectrometry for proteomic analysis (schematic shown in Figure 2). While this manuscript describes work we have employed for LAB, these protocols could also be used for the isolation of MVs from a variety of microbial species.

Figure 2. Schematic overview of the membrane vesicle isolation and characterization process. MVs are purified from Lactobacillus cultures initially via centrifugation and filtration, removing cells and large cellular debris. From the filtered supernatant, MVs are isolated by ultracentrifugation. Gradient centrifugation using OptiPrep medium can be applied to isolated MVs as an additional purification step. Purified MVs can then be characterized by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), SDS-PAGE protein gels, shotgun proteomics, or other methods.