A novel insertion sequence increases the expression of leukotoxicity in Actinobacillus actinomycetemcomitans clinical isolates

Background: The expression of leukotoxin varies among Actinobacillus actinomycetemcomitans strains and is dependent in part on the structure of the ltx promoter region. Highly leukotoxic strains, characterized by a 530 base pair (bp) deletion within the ltx promoter, have been associated with juvenile periodontitis in the United States and Europe. In the present study, we analyzed the ltx promoter structure to elucidate whether A. actinomycetemcomitans from Japanese periodontitis patients exhibits the highly toxic phenotype. Methods: Forty-five A. actinomycetemcomitans strains, including 43 clinical isolates, the highly leukotoxic strain JP2, and a minimally leukotoxic strain 652 were used in the study. The ltx promoter structure was analyzed by polymerase chain reaction (PCR), with oligonucleotide primers focusing the ltx promoter region, and nucleotide sequencing. Leukotoxic activity was determined by trypan blue exclusion. Western blotting assay was performed to detect the level of leukotoxin polypeptide. Results: A 495 bp PCR product was amplified from JP2, a 1025 bp product from 652 and 41 of the clinical isolates, and a 1926 bp product from the remaining two clinical isolates (Aa(IS)1, Aa(IS)2). Sequencing of the 1926 bp PCR fragment showed that it was similar to that of strain 652 but contained an 886 bp region that was identified as an insertion sequence (IS). Both Aa(IS) strains expressed high levels of leukotoxicity, similar to strain JP2. In addition, a mutant (Aa(IS-)) that had lost the IS element expressed a significantly lower level of leukotoxicity compared with Aa(IS) strains. Furthermore, the levels of leukotoxin polypeptide expressed by these strains were consistent with their whole cell leukotoxicity. Conclusions: A. actinomycetemcomitans clinical strains which were isolated from Japanese periodontitis patients do not possess the 530 bp ltx promoter deletion. The results of this study suggest that a high level of leukotoxin expression correlates with the insertion of the transposable DNA element.