Studying the fusogenic killing mechanism of hydramacin-1

The antimicrobial protein hydramacin-1 (HM-1) is an integral part of the immune defence of the Hydra, a fresh-water-dwelling metazoan. Its unique sequence, structure and killing mechanism have led to its classification in a new family of antimicrobial proteins (AMPS), the macins. It shows antimicrobial activity at μM concentrations against a wide range of bacteria, including multi-resistant human-pathogenic strains, making it relevant to future antimicrobial therapy. Previous results show that HM-1 promotes aggregation of bacteria using its two hydrophobic hemispheres, which surround an area of positive charge, however this mechanism is poorly characterised. HM-1 interactions with sparsely tethered bilayer lipid membranes (stBLM) were studied using neutron reflectometry in order to observe its fusogenic killing mechanism. stBLMs were formed by rapid solvent exchange using two common phospholipids, DMPC and negatively charged DMPG in a 3:1 ratio. HM-1 was observed interacting primarily with lipid headgroups, with limited binding to the outer lipid leaflet of the stBLM. Control experiments were performed where an stBLM was exposed to deuterated small unilamellar vesicles (SUVs), in which no meaningful increase in scattering length density (SLD) of the lipid membrane was observed. When this experiment was repeated in the presence of HM-1, a significant increase in SLD of the lipid membrane was detected with no evidence of membrane disruption, which would be observed in the case of a pore- forming AMP. This demonstrates that lipid molecules from the deuterated SUVs are incorporated into the lipid membrane, confirming the fusogenic mechanism of HM-1.