Two Adhesins Mediate Actinobacillus actinomycetemcomitans species-specific binding to BEC's

Objectives: Actinobacillus actinomycetemcomitans (Aa) possesses two outer membrane adhesins,Aae and ApiA that participate in binding to buccal epithelial cells (BEC). The purpose of this study was to examine the specificity of binding of the ApiA adhesin of Aa to BEC derived from a variety of mammalian species. Methods: We constructed a transposon mutant of Aa gene apiA in strain IDH781. The Aa mutant strain (IDH apiA-) and its wild type precursor strain IDH781 were mixed with human buccal epithelial cells for 1 hour at 37°C and BEC-bound-Aa was separated from unbound Aa. The BECs with Aa bound were plated on AaGM agar for determination of Aa per BECs and the ratio of Aa/BECwas calculated. We then expressed apiA in E. coli strain DH5a which conferred the ability of that E. coli strain to bind to BECs. We tested the ability of the constructed strain to bind to BECs derived from a variety of animal species including; cow, dog, horse, mouse, pig, sheep, old world and new world monkeys. We also constructed a double knockout strain (IDH aae-, apiA-) and tested it as well. Results: Binding to Human BECs was reduced from 426+9.9 to 21.4+6.3 (p<0.05 by Students unpaired one-tailed t test) when the wild type strain was compared to the apiA mutant strain. A plasmid harboring the wild-type apiA gene restored binding in the Aa mutant strain. E.coli expressing the ApiA adhesin showed a high degree of specificity and thus bound only to human and old world monkey BEC's. The double knockout strain of Aa showed no binding to BEC's from humans or old world monkeys. Conclusions: Results indicate that Aa expressing the ApiA and Aae adhesins show a high degree of specificity and thus bind only to BEC's in humans and old world monkeys.