(56d) Protein and Lipid Sorting on the Bacterial Membrane Regulate Bacterial Outer Membrane Heterogeneity

Gram-negative bacteria release nanoscale vesicles called outer membrane vesicles (OMVs) throughout growth to serve various functions, including delivery of toxins to both host and bacterial cells. Although the OMV is derived directly from the bacterial outer membrane (OM), numerous reports have demonstrated that the protein composition of the OMVs and OM can differ substantially. In particular, bacterial toxins are often reported to be enriched in the OMV relative to the OM. We undertook a series of studies to investigate the process by which a bacterial toxin, leukotoxin (LtxA), produced by the Gram-negative bacterium Aggregatibacter actinomycetemcomitans, is sorted to OMVs. This toxin is secreted via a one-step Type I secretion system across both the inner and outer membrane, but has been reported to re-associate with the bacterial OM. Our results demonstrate that LtxA is associated with the outer surface of the OMV and is enriched in the OMVs. We identified two populations of OMVs, one highly abundant population with diameters of approximately 100 nm, and a smaller population with diameters of approximately 350 nm. The small OMVs are produced throughout bacterial growth, while the large OMVs are produced only during later phases of growth. Interestingly, our results suggest that LtxA is only associated with the large OMVs. Because LtxA expression does not begin until later phases of growth, we hypothesized that LtxA itself might induce OM membrane curvature, thus promoting OMV budding. Instead, we found that variations in the lipopolysaccharide, present on the outer leaflet of the OM and OMV, drive both OMV formation and LtxA association with OMVs. Together, these results demonstrate a mechanism by which a bacterial protein (toxin) is sorted to secreted vesicles due to variations in its membrane affinity.