Mapping the epithelial-cell-binding domain of the Aggregatibacter actinomycetemcomitans autotransporter adhesin Aae

Abstract

The Gram-negative periodontopathogenAggregatibacter actinomycetemcomitans(Aa) binds selectively to buccal epithelial cells (BECs) of human and Old World primates by means of the outer-membrane autotransporter protein Aae. We speculated that the exposed N-terminal portion of the passenger domain of Aae would mediate binding to BECs. By using a series of plasmids that express full-length or truncated Aae proteins inEscherichia coli, we found that the BEC-binding domain of Aae was located in the N-terminal surface-exposed region of the protein, specifically in the region spanning amino acids 201–284 just upstream of the repeat region within the passenger domain. Peptides corresponding to amino acids 201–221, 222–238 and 201–240 were synthesized and tested for their ability to reduce Aae-mediated binding to BECs based on results obtained with truncated Aae proteins expressed inE. coli. BEC-binding ofE. coliexpressing Aae was reduced by as much as 50 % by pre-treatment of BECs with a 40-mer peptide (201–240; P40). Aae was also shown to mediate binding to cultured human epithelial keratinocytes (TW2.6), OBA9 and TERT, and endothelial (HUVEC) cells. Pre-treatment of epithelial cells with P40 resulted in a dose-dependent reduction in binding and reduced the binding of both full-length and truncated Aae proteins expressed inE. coli, as well as Aae expressed inAa. Fluorescently labelled P40 peptides reacted in a dose-dependent manner with BEC receptors. We propose that these proof-of-principle experiments demonstrate that peptides can be designed to interfere withAabinding mediated by host-cell receptors specific for Aae adhesins.