Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes.

Bacterial membrane vesicles (BMVs) are produced by all bacteria and facilitate a range of functions in host-microbe interactions and pathogenesis. Quantification of BMVs is a critical first step in the analysis of their biological and immunological functions. Historically, BMVs have been quantified by protein assay, which remains the preferred method of BMV quantification. However, recent studies have shown that BMV protein content can vary significantly between bacterial strains, growth conditions, and stages of bacterial growth, suggesting that protein concentration may not correlate directly with BMV quantity. Here, we show that the method used to quantify BMVs can alter experimental outcomes. We compared the enumeration of BMVs using different protein assays and nanoparticle tracking analysis (NTA). We show that different protein assays vary significantly in their quantification of BMVs and that their sensitivity varies when quantifying BMVs produced by different species. Moreover, stimulation of epithelial cells with an equivalent amount of BMV protein quantified using different protein assays resulted in significant differences in interleukin 8 (IL-8) responses. Quantification of Helicobacter pylori, Pseudomonas aeruginosa, and Staphylococcus aureus BMVs by NTA and normalization of BMV cargo to particle number revealed that BMV protein, DNA, and RNA contents were variable between strains and species and throughout bacterial growth. Differences in BMV-mediated activation of Toll-like receptors, NF-κB, and IL-8 responses were observed when stimulations were performed with equivalent BMV particle number but not equivalent protein amount. These findings reveal that the method of BMV quantification can significantly affect experimental outcomes, thereby potentially altering the observed biological functions of BMVs.