Dual function of the O-antigen WaaL ligase ofAggregatibacter actinomycetemcomitans

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, co-expression 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.

Regulation of adhesin synthesis in Aggregatibacter actinomycetemcomitans

“…The sequence has been found in all strains investigated and exists as two molecular weight forms dependent upon the serotype of the strain (Tang et al, 2007). In addition to the large size of the protein monomer, the adhesin is post-translationally modified using a shared mechanism involving the sugars (oligopolysaccharid) and enzymes (O-antigen ligase, or WaaL) associated with lipopolysaccharide biosynthesis (Danforth et al, 2022;Tang & Mintz, 2010;Tang et al, 2012). The functions of EmaA (collagen binding and biofilm formation) are conserved across A. actinomycetemcomitans serotypes (Danforth et al, 2019;Tang et al, 2007) and the energetic demands for synthesis, modification, and The study of the transcriptional regulation in A. actinomycetemcomitans, to date, has thus far been confined to a limited number of genes (Balashova et al, 2006;Brogan et al, 1994;Narayanan et al, 2017).…”

“…Serotype b emaA was expressed via the full‐length intergenic region using the previously described plasmid pKM09 (Ruiz et al., 2006; Table 1). The potential trans‐binding factors were expressed on an RK2 origin plasmid, which capably co‐replicates in the presence of pKM09 (Danforth et al., 2022). These RK2 origin plasmids were generated by PCR amplification of the P ltx :trans‐binding factor gene (described above) using a primer specific to the P ltx sequence (LtxpFSacI) and pKM2R (using a flanking SacI site from the plasmid multiple cloning site, Table 2).…”