Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin

Actinobacillus actinomycetemcomitans is associated with early onset periodontal diseases and secretes membranous vesicles that appear to contain several virulence-associated proteins. However, the composition of these vesicles and the process leading to their secretion are not well defined. Electron micrographs of thin sectioned bacterial cells and purified vesicle preparations showed that vesicles are spherical lipid bilayers, 50-100 nm in diameter, that appear to form by budding from the outer membrane of the bacterium. Thin layer chromatography identified the predominant lipid components of vesicles as lipopolysaccharide, phosphatidylethanolamine and cardiolipin, similar to the main lipid constituents of the outer membrane. However, vesicles also contained minor lipids that were not detected in outer membrane samples. The major protein constituents of vesicles co-migrated with proteins in outer membrane extracts of A. actinomycetemcomitans, but the outer membrane preparations possessed polypeptides that were not detected in vesicles. Three vesicle proteins were identified; the heat-modifiable OmpA homologue of A. actinomycetemcomitans, a 28 kDa lipoprotein related to the major outer membrane lipoprotein of Mannheimia haemolytica and leukotoxin. Incubation of leukotoxin-sensitive human HL60 cells with vesicles from A. actinomycetemcomitans strains JP2 and 652 resulted in cell lysis, indicating that vesicle-associated leukotoxin is biologically active. Vesicles from the highly leukotoxic strain JP2 were five- to 10-fold more toxic than vesicles from the minimally leukotoxic 652 strain. Furthermore, the specific leukotoxic activity of JP2 vesicles was approximately four- to five-fold higher than isolated outer membrane preparations from JP2, suggesting that vesicles are enriched in leukotoxin. Together, these results suggest that the formation of A. actinomycetemcomitans vesicles occurs by a process that results in the enrichment of leukotoxin.