Membrane Vesicles of Clostridioides difficile and Other Clostridial Species.

Membrane vesicles are secreted by growing bacterial cells and are important components of the bacterial secretome, with a role in delivering effector molecules that ultimately enable bacterial survival. Membrane vesicles of Clostridioides difficile likely contribute to pathogenicity and is a new area of research on which there is currently very limited information. This chapter summarizes the current knowledge on membrane vesicle formation, content, methods of characterization and functions in Clostridia and model Gram-positive species.

Production and purification of bacterial membrane vesicles for biotechnology applications: Challenges and opportunities.

Bacterial membrane vesicles (BMVs) are bi-layered nanostructures derived from Gram-negative and Gram-positive bacteria. Among other pathophysiological roles, BMVs are critical messengers in intercellular communication. As a result, BMVs are emerging as a promising technology for the development of numerous therapeutic applications. Despite the remarkable progress in unveiling BMV biology and functions in recent years, their successful isolation and purification have been limited. Several challenges related to vesicle purity, yield, and scalability severely hamper the further development of BMVs for biotechnology and clinical applications. This review focuses on the current technologies and methodologies used in BMV production and purification, such as ultracentrifugation, density-gradient centrifugation, size-exclusion chromatography, ultrafiltration, and precipitation. We also discuss the current challenges related to BMV isolation, large-scale production, storage, and stability that limit their application. More importantly, the present work explains the most recent strategies proposed for overcoming those challenges. Finally, we summarize the ongoing applications of BMVs in the biotechnological field.

Analysis of the utility of a rapid vesicle isolation method for clinical strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a pathogen capable of causing diseases ranging from mild to life-threatening, has a large arsenal of virulence factors. Notably, extracellular vesicles have emerged as significant players in the pathogenesis of this organism. However, the full range of their functions is still being studied, and difficulties related to vesicle purification (long protocols, low yields, and specialized instruments) have become a major obstacle for their characterization. In this context, the utility of rapid new methods of vesicle isolation from clinical strains is still unknown. Here, we analyze the utility of the ExoBacteria OMV isolation kit for a collection of clinical strains of P. aeruginosa. We first phenotypically characterized 15 P. aeruginosa strains to ensure that our samples were heterogeneous. We then determined the best conditions for purifying vesicles from P. aeruginosa PAO1 reference strain by the rapid method and used them to isolate vesicles from clinical strains. Our results indicated that M9 minimal medium is the best for obtaining high purity with the rapid isolation kit. Although we were able to isolate vesicles from at least four strains, the low yield and the large number of strains with unpurifiable vesicles showed that the kit was not practical or convenient for clinical strains. Our findings suggest that although fast procedures for vesicle purification can be of great utility for Escherichia coli, the more complex phenotypes of clinical isolates of P. aeruginosa are a challenge for these protocols and new alternatives/optimizations need to be developed.IMPORTANCEPseudomonas aeruginosa is recognized as an opportunistic pathogen in humans and animals. It can effectively colonize various environments thanks to a large set of virulence factors that include extracellular vesicles. Different methods were recently developed to reduce the time and effort associated with vesicle purification. However, the utility of rapid vesicle isolation methods for clinical strains of P. aeruginosa (which are recognized as being highly diverse) is not yet known. In this context, we analyzed the utility of the ExoBacteria OMV Isolation kit for vesicle purification in P. aeruginosa clinical strains. Our findings showed that the kit does not seem to be convenient for research on clinical strains due to low vesicle recovery. Our results underscore the importance of developing new rapid vesicle purification protocols/techniques for specific clinical phenotypes.

Single Extracellular Vesicle Imaging and Computational Analysis Identifies Inherent Architectural Heterogeneity.

Evaluating the heterogeneity of extracellular vesicles (EVs) is crucial for unraveling their complex actions and biodistribution. Here, we identify consistent architectural heterogeneity of EVs using cryogenic transmission electron microscopy (cryo-TEM), which has an inherent ability to image biological samples without harsh labeling methods while preserving their native conformation. Imaging EVs isolated using different methodologies from distinct sources, such as cancer cells, normal cells, immortalized cells, and body fluids, we identify a structural atlas of their dominantly consistent shapes. We identify EV architectural attributes by utilizing a segmentation neural network model. In total, 7,576 individual EVs were imaged and quantified by our computational pipeline. Across all 7,576 independent EVs, the average eccentricity was 0.5366 ± 0.2, and the average equivalent diameter was 132.43 ± 67 nm. The architectural heterogeneity was consistent across all sources of EVs, independent of purification techniques, and compromised of single spherical, rod-like or tubular, and double shapes. This study will serve as a reference foundation for high-resolution images of EVs and offer insights into their potential biological impact.

A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residues─which range in size and chemical composition─with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

Dataset of single nucleotide polymorphisms and comprehensive proteomic analysis of Streptococcus equi subsp. equi ATCC 39506.

Streptococcus equi subspecies equi (S. equi) is an opportunistic pathogen and a major causative agent of equine strangles, a contagious respiratory infection in horses and other equines. In this study, we provide the dataset associated with our research publication "Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infections" [1]. We describe the genomic differences between S. equi 4047 and S. equi ATCC 39506 and outline the comprehensive proteome information of various fractions, including the whole cell lysate, membrane proteome, secretory proteome, and extracellular vesicle proteome. In addition, we included a dataset of highly immunoreactive proteins identified through immunoprecipitation. The specifications table provides a detailed summary of the gene annotation and quantitative information obtained for each proteome. The proteomics data were analyzed using shotgun proteomics with LTQ Velos and Q Exactive mass spectrometry in the data-dependent acquisition mode. We have deposited the acquired data, including the mass spectrometry raw files and exported MASCOT search results, in the PRIDE public repository under the accession numbers PXD025152 and PXD025527.

Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential.

Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB+ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium.

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles.

Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32-72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8-43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.