Mapping the epithelial-cell-binding domain of the Aggregatibacter actinomycetemcomitans autotransporter adhesin Aae.
The Gram-negative periodontopathogen Aggregatibacter 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 in Escherichia 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 in E. coli. BEC-binding of E. coli expressing 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 in E. coli, as well as Aae expressed in Aa. 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 with Aa binding mediated by host-cell receptors specific for Aae adhesins.
Fine
Daniel H
DH
Department of Oral Biology, New Jersey Dental School, Newark, NJ 07103, USA.
Kaplan
Jeffrey B
JB
Department of Oral Biology, New Jersey Dental School, Newark, NJ 07103, USA.
Furgang
David
D
Department of Oral Biology, New Jersey Dental School, Newark, NJ 07103, USA.
Karched
Maribasappa
M
Department of Oral Biology, New Jersey Dental School, Newark, NJ 07103, USA.
Velliyagounder
Kabilan
K
Department of Oral Biology, New Jersey Dental School, Newark, NJ 07103, USA.
Yue
Gang
G
Department of Oral Biology, New Jersey Dental School, Newark, NJ 07103, USA.
D018829:Adhesins, Bacterial / Q000378:metabolism*; D001422:Bacterial Adhesion; D001665:Binding Sites; D002460:Cell Line; D004847:Epithelial Cells / Q000382:microbiology*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015661:Pasteurellaceae / Q000378:metabolism*; D010957:Plasmids
D018829:Adhesins, Bacterial