Separation of heterogeneous Aggregatibacter actinomycetemcomitans outer membrane vesicles reveals that toxin sorting is driven by surface-associated DNA

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with periodontal disease tend to produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is secreted via a type 1 secretion system, from the bacterial cytosol across both the inner and outer membranes in a single step into the supernatant, where it is able to interact with host cells. Upon secretion, some of the toxin associates with the bacterial cell membrane, enabling its release in association with the surface of outer membrane vesicles (OMVs), small, spherical vesicles derived from the outer membrane. We have previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs, a highly abundant population of small (<100 nm in diameter) OMVs, and a less abundant population of large (>300 nm in diameter) OMVs. Here, we have investigated the association of LtxA with the two populations of OMVs varying in size. Our results indicate that surface-associated DNA drives the selective sorting of LtxA to large OMVs.