Figure 6. Surface-exposed BilRI was expressed in various clinical isolates of A. actinomycetemcomitans grown in biofilms.

The surface exposure of BilRI in A. actinomycetemcomitans was examined via proteinase K treatment. During proteinase K treatment, protein expression was inhibited with chloramphenicol, and the maturation of the proprotein was inhibited with globomycin. Intact A. actinomycetemcomitans D7S cells were incubated with proteinase K for various time periods ranging from 0 to 24 hours, and the cells were then lysed via sonication. The samples were subsequently subjected to SDS-PAGE and immunoblotted with an anti-BilRI antibody. Proteinase K treatment decreased the amount of both forms of BilRI (red quadrangles) detected in outer membrane (OM) protein samples with two different antibody clones, 16B8 (A) and 16F7 (B), suggesting that BilRI was surface exposed in A. actinomycetemcomitans. To examine BilRI expression in the clinical isolates, cell lysates were obtained from young (21 hours) A. actinomycetemcomitans biofilm cultures through sonication. Samples containing approximately 0.3×10⁷ disrupted cells were subjected to SDS-PAGE and immunoblotted using an anti-BilRI antibody (B). The letters in parenthesis following the strain code indicate the serotypes (B).