Functional and Structural Analysis of EmaA of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a gram-negative facultative anaerobic bacterium associated with Localized Aggressive Periodontitis and cases of adult periodontitis. The bacterium colonizes the oral cavity and upper respiratory tract interacting with epithelial cells and extracellular matrix proteins. emaA (extracellular matrix protein adhesin A) encodes a 201 kDa outer membrane protein involved in adhesion of A. actinomycetemcomitans to collagen. 3 or 4 molecules of EmaA oligomerize to form antennae-like appendages on the bacterium surface. EmaA contains twelve putative collagen binding motifs. The first ten motifs are required for collagen binding and compose the ellipsoidal ending (head domain) of the EmaA structure. Collagen binding activity is dependent on the quarternary structure of EmaA. Objective: The objective of this study is to demonstrate the structure/function relationship of the twelve collagen binding motifs and collagen binding activity. Methods: emaA in-frame deletions were constructed by site directed mutagenesis and transformed into an emaA null mutant. Restoration of collagen binding activity was determined by ELISA. Surface appendages were visualized by transmission electron microscopy of phosphotungstic acid negatively stained whole mount specimens. The three-dimensional structures were reconstructed from double-axis tilt series of electron tomograms and image processing techniques. Results: Single motif deletion (motifs V-X) was constructed and the motifs essential for collagen binding within this region of the molecule were identified. Deletion of these sequences resulted in structural changes in the head domain of the surface appendages. A low resolution 3D structure was determined for the EmaA appendages on the surface of A. actinomycetemcomitans. Conclusions: Specific amino acid sequences of EmaA are associated with the collagen binding activity of A. actinomycetemcomitans. These sequences may play a structural role in the interaction of this pathogen with collagen fibers. This is the first structural investigation of a non-fimbrial adhesin of A. actinomycetemcomitans. This research was supported by NIH-NIDCR grant RO1-DE13824.