Characterization of Hybrids Made from Two Membrane Translocating Antimicrobial Peptides

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Antimicrobial peptides (AMPs) are a form of innate immunity in many organisms. They are promising substitutes for current antibiotics against which an increasing number of bacteria are becoming resistant. AMPs can form hybrids in which sequences of peptides are linked together by one or more amino acids, and these hybrids can potentially have increased activity. Most previous work on hybrids AMPs has involved peptides that kill bacteria by disrupting the cell membrane. However, here we focus on hybrid peptides that are synthesized from two peptides that translocate across the membrane without causing significant membrane disruption—the naturally occurring peptide buforin II (BF2) and the designed peptide DesHDAP1, both of which are derived from the core histone H2A. These peptides cross the cell membrane without disruption and bind to the target's nucleic acids to inhibit cellular function, killing the bacterium. We designed a series of BF2-DesHDAP1 hybrids aimed at systematically investigating the role of the sequence order and linkers on activity. Radial diffusion assays showed that although hybrid peptides with no linker or an alanine linker show little or no enhancement in activity compared to a mixture of the parent peptides, hybrid peptides with proline linkers were significantly more active. Interestingly, other bacterial assays showed that these hybrid peptides operate via a membrane-permeabilizing mechanism despite being derived from two peptides that cause little disruption on their own. We have also used circular dichroism (CD) spectroscopy to investigate how these activity trends relate to structure, finding that hybrids linked by proline actually exhibited increased helical secondary structure. Future work will utilize confocal microscopy to further investigate mechanisms of the hybrid peptides and test their cytotoxicity against human cells.