The
increase in antibiotic resistant bacteria is described as a major problem
facing health systems worldwide. Antimicrobial peptides (AMPs) have been
heralded as a novel antibiotic class as their membranolytic mechanism of action
does not readily induce resistance and are easily modified by solid-phase
peptide chemistry methods to alter/improve activity. Here we synthesise two
AMPs; magainin II and aurein 1.2 and substitute cationic amino acids with
unnatural amino acids or incorporate lipid moieties to mimic typical fatty acid
and quorum sensing molecules used in bacterial signalling. Using a microbial
flow cytometry assay to quantify AMP activity, we show that increasing peptide
concentration positively correlates with bacterial membrane disruption and that
amino acid substitution alters peptide specificity for Gram-negative and
Gram-positive bacteria. Further, the
incorporation of a fatty acid or 3-oxo-fatty acid at the N- or C-terminus of
aurein 1.2 has a dramatic effect on antimicrobial activity. Using outer membrane and cytoplasmic membrane
fluorescent based assays we show that for some analogues a loss in
antimicrobial activity does not correlate with a loss in bacteria-peptide
interaction. By using different bio-assays we have shown that amino acid
substitution or lipid incorporation have different effects on bacterial membrane
disruption, potential and interaction, thus pointing to the potential of these
assays in determining how a moiety may affect AMP activity.