Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans

Abstract

Actinobacillus actinomycetemcomitans is a Gram negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP), a rapidly progressing and severe disease of the oral cavity that affects predominantly adolescents. A. actinomycetemcomitans is also found in extraoral infections including infective endocarditis. As one of its many virulence determinants, A. actinomycetemcomitans produces the RTX (repeats in toxin) exotoxin, leukotoxin (LtxA). LtxA specifically kills leukocytes of humans and Old World Monkeys. All of our current knowledge of A. actinomycetemcomitans LtxA is based on the protein from strain JP2, a nonadherent laboratory isolate. Because laboratory isolates can lose virulence properties, we wished to examine LtxA from a clinical isolate, NJ4500. We show that localization patterns of LtxA do not differ between the strains. Subcellular localization studies with NJ4500 revealed that LtxA localizes to the outer membrane and that the interaction between LtxA and the surface of cells is specific. Surface localized LtxA was not removed with NaCl treatment and protease protection experiments revealed that approximately 10 kDa of LtxA is exposed. We purified secreted LtxA from NJ4500 and found that the specific activity of this toxin was greater than that of secreted LtxA from JP2. For other RTX toxins, fatty acid modification affects toxin activity, and A. actinomycetemcomitans LtxA is predicted to be modified. We show by two-dimensional gel electrophoresis that NJ4500 LtxA is more highly modified than JP2 LtxA, suggesting that the difference in activities could be due to differential modification. Studies of A. actinomycetemcomitans pathogenesis should therefore consider LtxA from clinical isolates.