Abstract
Outer membrane vesicles (OMVs) are nanosized spheres secreted by bacteria that are similar to the vesicles known as exosomes, which are secreted by most mammalian cell types. In contrast to many studies focusing on optimizing methods for enriching exosomes from biological fluid, few studies have been conducted to investigate outer membrane vesicles from fecal samples. Herein, we have developed a pipeline comprised of membrane filtration and multiple cycles of ultracentrifugation (UC) to isolate OMVs from fecal samples for proteomics analysis, where multiple cycles of UC are required for removal of contaminants. By iTRAQ labeling quantitative proteomics analysis, different filter sizes (0.22 μm and 0.45 μm) were compared in terms of their performance in enriching OMVs and eliminating background fecal material. Using the 0.45 μm filter, a slightly higher protein yield was obtained but no additional contaminating proteins from bacteria were identified compared to those from the 0.22 μm filter. The 0.45 μm filter together with the multiple cycles of UC were thus used to isolate OMVs for proteomics analysis. To our knowledge, this is the first study profiling a large number of OMV proteins from fecal samples. Such capabilities may help provide valuable information in understanding the communication between the host and microbiota, which is critical in preventing cancer and disease development
Highlights
Gut microbiota have the central role in maintaining intestinal homeostasis and the well-being of the host, which is regulated by communication between the host and microbiota
Due to the upper limit size of around 300 nm, it has been reported that processing the samples with a 0.22 μm filter may lead to a low yield of outer membrane vesicles (OMVs) that are captured by the filters, while using a 0.45 μm filter may result in contamination from the bacteria
In the images obtained from mouse stool, it should be noted that one cannot distinguish between mouse-derived extracellular vesicles versus bacterial OMVs
Summary
Gut microbiota have the central role in maintaining intestinal homeostasis and the well-being of the host, which is regulated by communication between the host and microbiota. Interaction between bacterial OMVs and the host modulates development and function of the immune system, which plays a critical role in disease and cancer development. The identification of specific protein components within OMVs may have great value for understanding the mechanism by which the OMVs interact with and regulate the innate immune responses in the host. Different size filters (0.22 μm and 0.45 μm) were compared for sample processing in terms of the performance in reducing contamination from bacteria and the improvement in OMV protein identification. On the basis of these comparisons, we developed an integrated pipeline to process fecal samples for OMV proteomic analysis (Figure 1). Such methodology is needed for future investigations of OMVs function in immune regulation and their roles in pathogenesis
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