Characterizing a Peptide Therapeutic Derived from the Cholesterol Recognition Amino Acid Consensus (CRAC) Motif of a Bacterial Toxin

Membrane cholesterol is a common target of pathogens, including bacteria and viruses, as well as bacterial toxins. Many of these pathogens exploit a cholesterol recognition amino acid consensus (CRAC) motif to facilitate cholesterol recognition. We have previously identified and characterized the CRAC motif used by a member of the repeats-in-toxin (RTX) family of toxins, the leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans, to bind to target cells. We hypothesized that a peptide consisting of the LtxA CRAC motif would bind strongly to cholesterol to inhibit the activity of LtxA. Using solid-phase peptide synthesis, we created a series of peptides based on the CRAC motif of LtxA and used a variety of techniques, including isothermal titration calorimetry, surface plasmon resonance and confocal microscopy, to measure the peptides’ affinity for cholesterol and ability to inhibit LtxA activity. Our wildtype CRAC peptide binds cholesterol with a comparable affinity as the toxin from which the sequence was derived (LtxA). This peptide specifically inhibits binding of LtxA to cholesterol and thus inhibits internalization and cytotoxicity of LtxA. A control peptide, in which the CRAC sequence has been scrambled, has a reduced affinity for cholesterol and does not inhibit the activity of the toxin, demonstrating the specificity of the interaction. A panel of peptides containing residue substitutions altering the peptide’s net charge demonstrated that the peptide’s charge significantly influences affinity for cholesterol and subsequent inhibition activity; highly charged peptides had no affinity for cholesterol, but surprisingly, a decreased charge did not enhance affinity. Previous simulations corroborated with our experimental results indicated the peptide resides on the surface of the membrane, and the peptide must maintain the proper balance of hydrophobic and hydrophilic character to retain activity. Importantly, the peptide does not affect cell viability over extended time periods, supporting its possible therapeutic use to inhibit the activity of cholesterol-binding pathogens. Although recognition of membrane cholesterol is a vital step in the mechanisms used by numerous pathogens, a method to inhibit this process without disrupting cellular function has not yet been uncovered. Here, we have designed a cholesterol-binding peptide for this purpose and demonstrated that it is effective in blocking bacterial toxin activity and is non-toxic to host cells.