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.

Comparison of Methods for Quantifying Extracellular Vesicles of Gram-Negative Bacteria.

There are a variety of methods employed by laboratories for quantifying extracellular vesicles isolated from bacteria. As a result, the ability to compare results across published studies can lead to questions regarding the suitability of methods and buffers for accurately quantifying these vesicles. Within the literature, there are several common methods for vesicle quantification. These include lipid quantification using the lipophilic dye FM 4-64, protein quantification using microBCA, Qubit, and NanoOrange assays, or direct vesicle enumeration using nanoparticle tracking analysis. In addition, various diluents and lysis buffers are also used to resuspend and treat vesicles. In this study, we directly compared the quantification of a bacterial outer membrane vesicle using several commonly used methods. We also tested the impact of different buffers, buffer age, lysis method, and vesicle diluent on vesicle quantification. The results showed that buffer age had no significant effect on vesicle quantification, but the lysis method impacted the reliability of measurements using Qubit and NanoOrange. The microBCA assay displayed the least variability in protein concentration values and was the most consistent, regardless of the buffer or diluent used. MicroBCA also demonstrated the strongest correlation to the NTA-determined particle number across a range of vesicle concentrations. Overall, these results indicate that with appropriate diluent and buffer choice, microBCA vs. NTA standard curves could be generated and the microBCA assay used to estimate the particle number when NTA instrumentation is not readily available.

Rapid quantification of vesicle concentration for DOPG/DOPC and Cardiolipin/DOPC mixed lipid systems of variable composition.

A novel approach to quantify mixed lipid systems is described. Traditional approaches to lipid vesicle quantification are time consuming, require large amounts of material and are destructive. We extend our recently described method for quantification of pure lipid systems to mixed lipid systems. The method only requires a UV-Vis spectrometer and does not destroy sample. Mie scattering data from absorbance measurements are used as input into a Matlab program to calculate the total vesicle concentration and the concentrations of each lipid in the mixed lipid system. The technique is fast and accurate, which is essential for analytical lipid binding experiments.

Rapid quantification of cardiolipin and DOPC lipid and vesicle concentration.

A novel approach to quantification of cardiolipin and DOPC lipid and vesicle concentration that is rapid and inexpensive is described. Traditional approaches to quantifying vesicle concentration destroy sample and are often time consuming. Using common laboratory equipment and software, lipid vesicles were reliably quantified allowing for immediate use without significant sample loss. Once calibrated, only absorbance measurements with a UV-Vis spectrophotometer are necessary as input into a Matlab program, which calculates the corresponding vesicle and lipid concentration. Fast and accurate concentration determination for preparations of vesicles is essential for analytical titration experiments necessary for protein/vesicle binding curves.

Bead-based flow-cytometry for semi-quantitative analysis of complex membrane vesicle populations released by bacteria and host cells.

During infection, the release of nano-sized membrane vesicle is a process which is common both for bacteria and host cells. Host cell-derived membrane vesicles can be involved in innate and adaptive immunity whereas bacterial membrane vesicles can contribute to bacterial pathogenicity. To study the contribution of both membrane vesicle populations during infection is highly complicated as most vesicles fall within a similar size range of 30-300nm. Specialized techniques for purification are required and often no single technique complies on its own. Moreover, techniques for vesicle quantification are either complicated to use or do not distinguish between host cell-derived and bacterial membrane vesicle subpopulations. Here we demonstrate a bead-based platform that allows a semi-quantitatively analysis by flow-cytometry of bacterial and host-cell derived membrane vesicles. We show this method can be used to study heterogeneous and complex vesicle populations composed of bacterial and host-cell membrane vesicles. The easy accessible design of the protocol makes it also highly suitable for screening procedures to assess how intrinsic and environmental factors affect vesicle release.