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

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.