Characterization and Analysis of Leukotoxin-containing Outer Membrane Vesicles

Abstract

Outer membrane vesicles (OMVs) are nanoscale, membrane-bound structures that bleb off from the outer membrane of Gram negative bacteria. Although being discovered over 30 years ago, only in the last decade have OMVs garnered attention for their bacterial function. Specifically, they have been shown to be enriched in bacterial toxins as well as other virulence factors, but not all OMVs in a single population will have the same content. Heterogeneity amongst OMV populations, in terms of size, function and composition, has served as a point of debate within the field, resulting in the inability to reach a consensus on important OMV mechanisms. Heterogeneity has often been rejected due to limited analyses. In this work, we have captured leukotoxin-containing OMVs, produced by Aggregatibacter actinomycetemcomitans (Aa), through modifying a quartz crystal microbalance with dissipation monitoring (QCM-D) sensor with an anti-leukotoxin antibody. Our previous work demonstrated that there are two distinct populations of Aa OMVs, one smaller (<100 nm) and one larger (∼300-350 nm) and that leukotoxin is preferentially sorted to the larger OMVs. To further examine this concept, a polymer array with conjugated anti-leukotoxin antibody was assembled on a QCM-D sensor. OMVs were injected over the antibody-functionalized sensor, exclusively capturing the larger OMVs. Fluorescence imaging of individual OMVs also confirms this size heterogeneity based on toxin content. This application provides a biosensing strategy to differentiate OMV subpopulations, especially with respect to the distribution of toxin content as a function of OMV size.