Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innate immunity against pathogens in insects. The production of AMPs is costly owing to resource-based trade-offs, and strategies maximizing the efficacy of AMPs at low concentrations are therefore likely to be advantageous. Here, we show the potentiating functional interaction of co-occurring insect AMPs (the bumblebee linear peptides hymenoptaecin and abaecin) resulting in more potent antimicrobial effects at low concentrations. Abaecin displayed no detectable activity against Escherichia coli when tested alone at concentrations of up to 200 μM, whereas hymenoptaecin affected bacterial cell growth and viability but only at concentrations greater than 2 μM. In combination, as little as 1.25 μM abaecin enhanced the bactericidal effects of hymenoptaecin. To understand these potentiating functional interactions, we investigated their mechanisms of action using atomic force microscopy and fluorescence resonance energy transfer-based quenching assays. Abaecin was found to reduce the minimal inhibitory concentration of hymenoptaecin and to interact with the bacterial chaperone DnaK (an evolutionarily conserved central organizer of the bacterial chaperone network) when the membrane was compromised by hymenoptaecin. These naturally occurring potentiating interactions suggest that combinations of AMPs could be used therapeutically against Gram-negative bacterial pathogens that have acquired resistance to common antibiotics.