Identification of Receptor-Based Peptides to Inhibit Leukotoxin Activity

Abstract

The leukotoxin (LtxA), secreted by the bacterium Aggregatibacter actinomycetemcomitans, is a member of the RTX family of toxins which targets leukocytes through several mechanisms. The toxin's specificity for human white blood cells is driven by its recognition of the lymphocyte function-associated antigen 1 (LFA-1) integrin. This project investigates inhibition of LtxA-LFA-1 binding on living cells as an anti-virulence strategy to inhibit LtxA cytotoxicity. Specifically, we are investigating using small peptides to block (A) LFA-1 binding sites on LtxA and (B) LtxA-binding sites on LFA-1. Several LtxA binding sites on LFA-1, including a region on the αL β-propeller, have been proposed. Therefore, we have synthesized peptides corresponding to these domains and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxicity in human immune cells. We found that four of the five peptides, specifically those corresponding to sequential β-strands in the β-propeller domain, inhibited LtxA activity, demonstrating the effectiveness of this approach. The LFA-1 binding sites on LtxA are less established, but three monoclonal antibodies have previously been identified based on their inhibition of LtxA activity. We hypothesized that the epitopes to at least some of the antibodies are involved in this essential recognition process and synthesized peptides based on these epitopes to block the LtxA binding site on LFA-1. We are currently applying biophysical methods to investigate the mechanism by which these peptides inhibit LtxA binding to LFA-1 to fully characterize this protein-protein interaction. The results of our investigations will facilitate the development of peptides to inhibit LtxA activity and can have broader implications through applying a similar approach to hinder the cytotoxic activity of other RTX toxins.