Aurein 1.2 is a short, 13 residue, cationic peptide derived from the secretions of Litoria raniformis, shown to have moderate antimicrobial activity. Amino acid substitution is known to effect antimicrobial peptide activity, particularly by increasing the hydrophobic or cationic characteristics of a peptide. Replacing residues in a peptide can alter secondary and tertiary structure and arrangement of hydrophobic, hydrophilic and amphipathic regions. Aurein 1.2 contains N- and C-terminal regions anionic residues, aspartic acid and glutamic acid respectively. Our study substituted one or both of these residues with cationic (Lys or Arg) or hydrophobic (Phe) residues and determined the effect on antimicrobial activity. By substituting Asp or Glu with cationic amino acids, antimicrobial activity was improved for both Gram-positive and Gram-negative bacteria. The effect is also cumulative, with substitution of both providing greater activity than either substitution alone. We show that aurein 1.2 is not a good candidate for modification into a proline-rich peptide. We performed a ‘proline’ scan which resulted in worse activity that native aurein 1.2. This is likely because modelling shows that it does not develop the signature ‘proline kink’ associate with strong antimicrobial activity in other peptides with proline residues such as maculatin.
A major hurdle in antimicrobial peptide development is translation to clinical use, where often the AMP performs worse than in pre-clinical testing. A probable factor is the assay buffer used in preclinical testing being significantly different from body or tissue fluid where the AMP is used. To investigate the effect of assay media we used a range of typically used media and a developed a simulated body fluid. . Our experiments show that when using simulated body fluid, in vitro antimicrobial testing is more accurate. Using bacterial flow cytometry, the effects of typical test media on peptide activity and bacteria can be visualised.