Search Results (3)

Bacterial outer membrane vesicles (OMVs) are nanoscale extracellular structures produced by Gram-negative bacteria that are critical for microbial biology and host-pathogen interactions and have great potential in biotechnological applications. Despite the availability of fluorescent dyes for OMV studies, many are repurposed from eukaryotic extracellular vesicle research and are not explicitly optimized for OMVs, leading to challenges in achieving consistent labeling, minimizing background noise, and preserving vesicle integrity during analyses. This study evaluates B2, a benzimidazole-derived fluorophore, for OMV labeling in advanced techniques like flow cytometry and confocal microscopy. OMVs were isolated from Escherichia coli strains BL21 and O157, and their integrity was confirmed using transmission electron microscopy (TEM). B2 staining protocols were optimized for OMVs, and fluorescence analyses revealed specific interactions with the vesicle membranes, reducing aggregation and enhancing signal uniformity. Flow cytometry indicated near-complete labeling efficiency (98–100%) with minimal background interference. Confocal microscopy further validated B2’s effectiveness, showing evident OMV internalization into epithelial HT-29 cells and compatibility with other fluorophores. Density functional theory (DFT) calculations, including Fukui function analysis, identified key electrophilic and nucleophilic regions in B2 that facilitate specific hydrogen bonding and polar interactions with membrane components. Non-covalent interaction (NCI) analysis revealed pronounced intramolecular hydrogen bonding along with discrete regions of weak van der Waals interactions. Molecular dynamics simulations suggest that B2 exhibits an affinity for both the hydrophobic core of the lipid bilayer and the core oligosaccharide region of the LPS layer, which collectively ensures sustained retention of the dye. The findings presented in this study position B2 as a valuable fluorophore for OMV research. Full article

Extracellular vesicles (EVs) are critical elements of cell–cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation. Full article

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQD_S._b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQD_S._b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQD_S._b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQD_S._b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme. Full article