Identifying size-dependent toxin sorting in bacterial outer membrane vesicles

Abstract

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. Despite being isolated from a single population of bacteria, OMVs can exhibit heterogeneous size and toxin content, which can be obscured by assays that measure ensemble properties. To address this issue, we utilize fluorescence imaging of individual OMVs to reveal size-dependent toxin sorting. Our results showed that the oral bacterium Aggregatibacter actinomycetemcomitans (A.a.) produces OMVs with a bimodal size distribution, where larger OMVs were much more likely to possess leukotoxin (LtxA). Among the smallest OMVs (< 100 nm diameter), the fraction that are toxin positive ranges from 0-30%, while the largest OMVs (> 200 nm diameter) are between 70-100% toxin positive. Our single OMV imaging method provides a non-invasive way to observe OMV surface heterogeneity at the nanoscale level and determine size-based heterogeneities without the need for OMV fraction separation.

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

“…Our observations that OMVs produced during the late exponential phase contain more LtxA and consist of a greater fraction of large OMVs are consistent with our prior finding that LtxA is more abundant on large OMVs [ 39 , 40 , 41 ]. Knowing that LtxA is located on the surface of OMVs [ 12 ] and that it is a membrane-active protein that is able to induce curvature in model membrane systems [ 42 ], we hypothesized that the preferential association of LtxA with large OMVs might be mediated by the production of LtxA during the exponential phase.…”

“…We tested both conditions by first pretreating JP2 OMVs collected from the late exponential phase with PMB or DNase I and measuring the resulting changes in the LtxA elution profile by size exclusion chromatography (SEC). We have previously shown that this SEC approach is able to separate large and small A. actinomycetemcomitans OMVs [ 39 , 41 ]; thus, we anticipated that a similar approach could be used to detect variations in the LtxA composition of large and small OMVs after PMB or DNase I treatment.…”

Outer Membrane Vesicles Released from Garlic Exosome‐like Nanoparticles (GaELNs) Train Gut Bacteria that Reverses Type 2 Diabetes via the Gut‐Brain Axis

“…[53][54][55] OMVs, being extracellular nanovesicles released by bacteria, exhibit heterogeneity in size (40-300 nm diameter), [11,[56][57][58][59][60] where the size dictates both the entry into host cells and the protein composition. [61][62][63] In our investigation, we noted that a fraction of orally administered A. muciniphila OMVs evades uptake during the journey from the gut to the brain, escaping uptake by other cell types and ultimately being taken up by microglial cells. We propose that a specific subset of OMVs possesses both an "eat me" signal tailored for brain microglial cells and a "do not eat me" signal that is universal for resident cells such as macrophages in other tissues.…”

“…Nevertheless, recent exciting advances have been made possible in understanding the heterogeneous nature of secreted nanoparticles including OMVs, and defining OMVs subpopulations. [52][53][54]63] Therefore, our findings provide a rationale for the isolation and characterization of subsets of OMVs and further elucidation of the precise role of each subset of OMVs to determine whether they carry both an "eat me" signal tailored for brain microglial cells and a "do not eat me" signal that is universal for resident cells in other tissues in both physiological and pathophysiological processes.…”

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

“…However, there have been numerous reports of its resistance to commonly used antibiotics for treatment 43,44 . As part of its virulence, A. actinomycetemcomitans produces leukotoxin (LtxA), which is secreted into the extracellular milieu as a free protein and is also released in association with OMVs, where it resides on the surface along with surface-associated DNA [45][46][47] . This highlights the critical role of OMVs in pathogenesis, which is why we seek to utilize them, given their relevance in the disease process.…”