Single Amino Acid Changes Impact the Ability of <i>Drosophila melanogaster</i> Cecropins to Inhibit Growth of <i>Providencia</i> Pathogens

As antibiotic-resistant bacteria spread worldwide, the need to develop novel antimicrobial agents is urgent. One rich source of potential antimicrobials is the insect immune system, as insects produce a wide range of antimicrobial peptides (AMPs) with diverse sequences and structures. Insects also encounter many bacterial pathogens, some of which are closely related to pathogens of clinical relevance. However, despite interest in AMPs as therapeutics, the relationships between the amino acid sequence, biophysical properties, antimicrobial activity, and specificity are still not generalizable. To improve our understanding of these relationships, we assessed how single amino acid changes in cecropin AMPs produced by the fruit fly, <i>Drosophila melanogaster</i>, impact both their structure and their ability to inhibit the growth of <i>Providencia</i> species isolated from wild-caught <i>D. melanogaster</i>. These pathogens are of particular interest as they have a range of virulence in fruit flies, and work <i>in vivo</i> suggests that differences in virulence could be partially attributable to differential susceptibility to AMPs. <i>D. melanogaster</i> cecropins are 40 amino acids long but vary at only 5 residues with largely conservative changes. We found that these changes could impact inhibitory concentrations by up to 8-fold against <i>Providencia</i> species. Our investigation focused on a single amino acid position due to the importance of a flexible "hinge" in cecropin function. We found that altering the identity of this amino acid alone greatly impacted antimicrobial activity, changing bacterial susceptibility up to 16-fold. Generally, <i>Providencia</i> species that are less virulent <i>in vivo</i> are more susceptible to cecropin AMPs <i>in vitro</i>. We also observed differences in the kinetics of permeabilization and bacterial killing between species, suggesting that peptide-membrane interactions were differently affected by single amino acid changes and that bacteria in this genus may vary in their membrane composition.