Abstract
Protein glycosylation is critical to the quaternary structure and collagen-binding activity of the extracellular matrix protein adhesin A (EmaA) associated with Aggregatibacter actinomycetemcomitans. The glycosylation of this large, trimeric autotransporter adhesin is postulated to be mediated by WaaL, an enzyme with the canonical function to ligate the O-polysaccharide (O-PS) antigen with a terminal sugar of the lipid A-core oligosaccharide of lipopolysaccharide (LPS). In this study, we have determined that the Escherichia coli waaL ortholog (rflA) does not restore collagen binding of a waaL mutant strain of A. actinomycetemcomitans but does restore O-PS ligase activity following transformation of a plasmid expressing waaL. Therefore, a heterologous E. coli expression system was developed constituted of two independently replicating plasmids expressing either waaL or emaA of A. actinomycetemcomitans to directly demonstrate the necessity of ligase activity for EmaA collagen binding. Proper expression of the protein encoded by each plasmid was characterized, and the individually transformed strains did not promote collagen binding. However, coexpression of the two plasmids resulted in a strain with a significant increase in collagen binding activity and a change in the biochemical properties of the protein. These results provide additional data supporting the novel hypothesis that the WaaL ligase of A. actinomycetemcomitans shares a dual role as a ligase in LPS biosynthesis and is required for collagen binding activity of EmaA.