Comparative ultrastructure of leukotoxic and non‐leukotoxic strains of Actinobacillus actinomycetemcomitans

Fourteen strains of Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) have been examined by transmission electron microscopy. The cells, which grow as coccobacillary forms on agar medium, have a cell periphery ultrastructurally compatible with that of other Gram-negative species. However, the cell membrane and peptidoglycan layer are not readily visualized following standard processing. Of particular interest was the finding of numerous extracellular membranous vesicles in A. actinomycetemcomitans strains known to have leukotoxic activity, while non-leukotoxic strains demonstrated few or no extracellular vesicles. Similar findings were noted whether cells were grown on blood agar plates or in thioglycollate broth. Antisera were obtained in rabbits against whole cells of leukotoxic strain Y4. The resulting antisera successfully labeled vesicles associated with 4 separate leukotoxic strains. The label was completely retained on the vesicles after absorption of the antisera with non-leukotoxic strain ATCC 29523. Non-leukotoxic strains showed some patchy surface labeling with non-absorbed antiserum to Y4 and no labeling after absorption with ATCC strain 29523. These observations suggest that extracellular membranous vesicles are associated with leukotoxic activity and may serve as a useful source of leukotoxic factor. These conclusions are strengthened by the observation of a strong correlation between the concentration of extracellular vesicles in strains grown on agar and their relative leukotoxicity as reported by other investigators.