In Depth Analysis of the Metal Binding Interaction and Mode of Action of the Antimicrobial Peptide Gaduscidin-1

Antibiotic resistance has been accelerating in the past decades, as the implementation of new antibiotic compounds has decreased drastically. Antimicrobial peptides (AMPs) are well studied, broad-spectrum therapeutic agents that may be a novel approach to combat antibiotic resistance. AMPs are found ubiquitously in nature and are used as part of an organisms innate immune system. They have evolved overtime to be an essential part of the host immune defense against pathogens. Here, the in depth examination of the AMP Gaduscidin-1 or Gad-1 was investigated to explore the metal binding advantages and mode of action of Gad-1. The mode of action of Gad-1 against Gram-positive bacteria at varied pH conditions was investigated and revealed the possibility that the major mode of action may differ at neutral and low pH. Using SYTOX green uptake assays and confocal microscopy it was shown that Gad-1 is more rapidly internalized at low pH, while at neutral pH there was lesser internalization and was primarily membrane bound in confocal experiments. This and other evidence point to the possibility that Gad-1 may have different modes of action at pH 7.0 and pH 5.5. Further, the metal binding ability and pH independent activity of Gad-1 was exploited to treat Pseudomonas aeruginosa biofilms in an environment that mimic the disease cystic fibrosis. The unique nuclease activity of Gad-1 is able to cleave extracellular DNA, an essential component of biofilms, to help destabilize P. aeruginosa biofilms. This coupled with the bactericidal action of Gad-1 contributed to the eradication of both planktonic and biofilms of P. aeruginosa. Moreover, Gad-1 could be used in combination with kanamycin to increase biofilm clearance through synergistic interactions. Overall, the mode of action of Gad-1 was further elucidated and a medical application for Gad-1 was investigated and shown to be promising in preliminary experiments, showing the power of AMPs as therapeutic agents.