Identification and Characterization of adhesins from Prevotella intermedia 17

Objectives: Prevotella intermedia binds and invades variety of host cells. This binding is most probably mediated through cell surface proteins termed adhesins. P. intermedia 17 genome contains several genes encoding putative proteins similar to adhesins from other bacteria. We characterized genes encoding proteins homologous to the Tannerella forsythensis BspA adhesin.

Methods: Genes encoding three homologs of the BspA protein were cloned and expressed in Escherichia coli. Recombinant BspA-like proteins were examined for their ability to bind to extracellular matrix proteins using ELISA. The adhesive/invasive function was also further analyzed using proteins expressed in heterologous host E. coli.

Results: The first analyzed gene encoded protein that shared 25% sequence identity with BspA-surface antigen of T. forsythensis (gb AAC82625.1). The protein also showed significant similarity with hypothetical proteins of Bacteroides fragilis (dbj BAD48495.1) and hypothetical protein of Bacteroides thetaiotaomicron (gb AA076347.1). ELISA analysis indicated that this protein can bind multiple ECM-proteins: fibrinogen, fibronectin and collagen. Interestingly, E. coli expressing the protein was highly invasive in both mouse NIH3T3 fibroblasts as well as in Human Umbilical Cord Endothelial Cells when compared to E. coli containing the expression vector only thus indicating this protein plays a major role in invasion of P. intermedia 17 into variety of host cells. Two other genes, PI1564 and PI1571, encoding proteins with similarity to the BspA adhesin of T. forsythensis also bound ECM-like proteins. Our ELISA results showed the PI1564-encoded protein binds specifically to collagen and the PI1571-coded protein binds to fibrinogen. These results were confirmed using competitive ELISA studies.

Conclusions: P. intermedia 17 contains multiple adhesins belonging to the family of BspA-like proteins. Despite sequence similarity these proteins specifically bind to different host components thus mediating the attachment or invasion processes of the bacterium, respectively.

This study was supported by an NIH grant R01DE016124 to J.P. Lewis.