(191br) The Role of Bacterial Outer Membrane Vesicles in Establishing an Ecological Niche

Gram negative bacteria constitutively secrete outer membrane vesicles (OMVs), which are small, membrane-bound, spherical vesicles that encapsulate a number of molecules including toxins and other virulence factors. These vesicles are hypothesized to play a role in the establishment of an ecological niche for the organism by delivering cargo to both eukaryotic and prokaryotic cells. In this project, we have studied the role of OMVs in the ability of an oral pathogen, Aggregatibacter actinomycetemcomitans, to colonize the host, by communicating with host cells as well as neighboring bacteria. Using dynamic light scattering (DLS) and scanning electron microscopy (SEM), we found that A. actinomycetemcomitans OMVs are approximately 85 nm in diameter and enriched in a protein toxin, leukotoxin (LtxA). LtxA is located entirely on the surface of the vesicle, bound to the negatively charged lipopolysaccharide (LPS) present on the surface of the vesicle through electrostatic interactions. We hypothesized that in this location, LtxA might act as a targeting agent, trafficking the OMVs to target cells in a cholesterol- and receptor-dependent manner. Because free LtxA must bind to both cholesterol and a specific receptor, lymphocyte function-associated antigen-1 (LFA-1) in order to kill target cells, we investigated the specific role of these two molecules in the binding and toxicity of A. actinomycetemcomitans OMVs. Our results, however, demonstrate that A. actinomycetemcomitans OMVs require neither cholesterol nor LFA-1 to induce toxicity. Additionally, we have discovered that unlike free LtxA, OMVs are not endocytosed by target cells; instead they fuse with the plasma membrane of the host cell to deliver their cargo directly into the cytosol. We have also characterized delivery of A. actinomycetemcomitans OMVs to other A. actinomycetemcomitans cells and found that the OMVs are efficiently taken up by the bacterial cells. The presence of OMVs appears to decrease the extent of bacterial growth, consistent with reports of OMVs containing quorum sensing molecules. We are currently investigating the delivery of OMVs to other periodontal inhabitants to study the role of OMVs in the establishment of the oral microbiome and biofilms. We anticipate that these results will facilitate the identification of new therapeutic targets aimed at preventing pathogen colonization within the oral microbiome.