Molecular heterogeneity of EmaA, an oligomeric autotransporter adhesin of Aggregatibacter (Actinobacillus) actinomycetemcomitans

Abstract

Adhesion of Aggregatibacter actinomycetemcomitans to extracellular matrix proteins is mediated by antennae-like surface structures composed of EmaA oligomers. EmaA is an outer-membrane protein orthologous to the autotransporter YadA, a virulence determinant of Yersinia. emaA was present in the 27 strains examined, covering the six serotypes of A. actinomycetemcomitans. Ten individual genotypes and three different forms of the protein (full-length, intermediate and truncated) were predicted. The prototypic, full-length EmaA (202 kDa) was only associated with serotypes b and c, which displayed antennae-like surface structures. These strains bound to collagen embedded in a 3D matrix. The intermediate form of EmaA (173 kDa) was exclusively associated with serotypes d and a, which contained a 279 aa in-frame deletion, as well as a different N-terminal head domain sequence. These differences modified the appearance of the EmaA structures on the cell surface but maintained collagen-binding activity. Strains containing the truncated form of EmaA had single or multiple substitutions, deletions or insertions in the sequences, which resulted in the absence of EmaA molecules on the outer membrane and loss of collagen-binding activity. Population structure analyses of this organism, based on emaA, indicated that serotypes b and c belonged to one subpopulation, which was independent of the other serotypes. The main divergence was found in the functional head domain. The conserved emaA genotype within serotypes suggests a stable clonal linkage between this autotransporter protein and other virulence determinants.

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…Both pathways are distinguished later by the difference in specificities and active sites of the competing enzymes Wzy, WaaL, and PilO (390). The EmaA adhesin of A. actinomycetemcomitans is modified with O-antigen sugars and is a substrate of the WaaL enzyme (437)(438)(439). The same is true for B. pseudomallei, where the glycan attached to the RgpA glycoprotein is similar to the anionic polysaccharides (428,431).…”

“…This protein trimerizes to form antenna-like structures that can bind collagen. EmaA is modified with the O PS moiety of LPS containing fucose, rhamnose, and GalNAc (437,438). Strikingly, the O PS is attached in the periplasm to the protein via the activity of the O-antigen ligase WaaL (437,439).…”

Glycosylation of the Collagen Adhesin EmaA ofAggregatibacter actinomycetemcomitansIs Dependent upon the Lipopolysaccharide Biosynthetic Pathway

“…Equivalent amounts of membrane protein, determined by absorbance at 280 nm, were prepared in the loading buffer as described by the immunoblotting of LPS and loaded into 4 to 15% polyacrylamide Tris-HCl gels. Electrophoresis was performed at 40 V and 4°C for 15 h. The separated proteins were transferred to an Optitran nitrocellulose membrane at 70 V and 4°C for 105 min and probed with an anti-EmaA monoclonal antibody (46). The immune complex was detected using HRP-conjugated goat anti-mouse IgG (Jackson Laboratory, Bar Harbor, ME) and visualized as described by the immunoblotting of LPS.…”

“…The membrane protein fraction of A. actinomycetemcomitans was prepared as described previously (24,46). Briefly, 200 ml late logarithmic phase cells were harvested and resuspended in 2.5 ml of 10 mM HEPES (pH 7.4) with 1 mM phenylmethylsulfonyl fluoride (PMSF; USB Corporation, Cleveland, OH) and 1ϫ complete protease inhibitor cocktail (Roche Diagnostic Corporation, Atlanta, GA).…”

“…Our earlier work suggested a correlation between the type of LPS molecule and the form of EmaA synthesized by A. actinomycetemcomitans (46). The EmaA of serotype b A. actinomycetemcomitans is a 202-kDa protein that forms the antennae-like appendages found on the surface of A. actinomycetemcomitans and is required for collagen binding (40).…”