Pathogenic bacteria produce numerous virulence factors, including protein toxins, to enhance their growth and survival within the host. Our lab seeks to understand the mechanisms of bacterial toxin delivery to identify and exploit therapeutic targets. In this project, we have focused on a member of the repeats-in-toxin (RTX) family of proteins, which are secreted by many Gram negative bacteria, including Bordetella pertussis, Escherichia coli, and Aggregatibacter actinomycetemcomitans. The leukotoxin (LtxA) secreted by A. actinomycetemcomitans specifically kills human white blood cells to disrupt the host immune response and therefore plays a key role in bacterial colonization of the host. The cell type specificity of LtxA arises from its reported targeting of the lymphocyte function-associated antigen-1 (LFA-1) integrin, which is only expressed by human white blood cells. In addition, we recently showed that LtxA binds with a strong affinity to cholesterol, and inhibition of this binding to cholesterol prevents LtxA-mediated cytotoxicity. Our goal in this project was to use information about these two targets of LtxA to design and test a panel of anti-toxin peptides, as an anti-virulence strategy to inhibit the pathogenesis of A. actinomycetemcomitans. (A) Cholesterol. We have identified a cholesterol recognition amino acid consensus (CRAC) motif in the toxin that regulates the interaction of the toxin with cholesterol. Using this CRAC sequence, we designed a cholesterol-binding peptide that inhibits LtxA-mediated cytotoxicity of human immune cells by blocking the binding of the toxin to cholesterol. (B) LFA-1. Using an analogous approach, we synthesized peptides corresponding to the hypothesized binding domains on the β-propeller of the αL subunit of LFA-1 and characterized their capability to inhibit LtxA binding and subsequent cytotoxicity. We found that four of the five peptides, specifically those corresponding to sequential β-strands in the β-propeller domain, inhibited LtxA activity. We are currently applying biochemical methods to investigate the mechanism by which these peptides inhibit LtxA binding to its targets to fully characterize the inhibitory mechanisms of these peptides. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.

Inhibition of bacterial toxin activity using receptor-based peptides

255th National Meeting (2018)

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