Bacterial Extracellular Vesicles: a new target for a diagnostic device

Abstract

Extracellular vesicles are produced by all the species across the domains of life, suggesting that vesiculation represents a fundamental principle of living matter, and over the years, the study of bacterial extracellular vesicles (bEVs) has gained more attention regarding their roles in bacterial survival, interactions with other cells and pathogenesis. The first aim of my study was to isolate and characterize bEVs from Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). I found that using Size Exclusion Chromatography (SEC) technique, the bEVs at fractions 2-5 gave highest particle counts enriched with protein contents for E. coli and S. aureus during characterization with Nanosight Tracking Analysis (NTA) with size mean of 50-200 nm. Moreover, the structure of bEVs was confirmed with Transmission Electron Microscopical (TEM) showing regular spherical double membranes structures of a size range 50-100nm. Western blotting with Anti-LPS antibody detected LPS bands in both cell lysate and EVs of E. coli UPEC 536. Results of the dissociation constant (Kd) of different concentrations of single strand DNA aptamer (GN6-Cy5) aptamer achieved binding affinity towards E. coli DH5α, Nissle 1917, MG 1655 and UPEC 536 at 223.4, 325.5, 320.2 and 300.4 nM, respectively based on detecting the fluorescence intensity. This was confirmed by Stimulated Emission Depletion (STED) Microscopy during examining the GN6 aptamer and some red spots were detected around the surfaces of E. coli UPEC 536 bacteria. Recently a new dye was introduced to our Lab for labelling the bEVs called Acoerela (Aco), I applied the Aco-600 to dye the UPEC 536 EVs. Despite optimizing the ideal concentration of less to nil detected noise in the system background for theAco-600, UPEC 653 EVs and GN6-Cy5 aptamer, results of ZetaVeiw fluorescence analysis using 488nm laser didn’t detect any signal for labeling UPEC 536 EVs with the Aco-600, neither during incubating the UPEC 536 with GN6-Cy5 aptamer. The STED images for labelling UPEC 536-EVs with Aco-600 dye showed orange-stained spherical structures of EVs of a size around 100-150 nm. While no co-localization was detected from STED nor confocal microscopy images after incubation of the labelled UPEC 536 EVs Aco-600 mixture with the GN6-Cy5 aptamer.