Suicide probes: a resource to assess the mechanism of action of membrane-active peptides in living bacterial cells.

DUPUY, FERNANDO G.; CHALÓN, MIRIAM CAROLINA; RIOS COLOMBO, NATALIA SOLEDAD; BELLOMIO, AUGUSTO

Jornada; Jornadas Virtuales YIB Talks; 2020

Young Initiative on Biophysics (YIB) - International Union of Pure and Applied Biophysics (IUPAB)

Class II bacteriocins are membrane-active peptides that have antimicrobial activity against pathogenic bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to bacterial membrane permeabilization. However, little is known about the events triggering the pore formation after the bacteriocin recognizes the receptor. There are two theoretical models. The first one suggests that the pore is the receptor forced into an open conformation by the bacteriocin. In the second model, the receptor acts as an anchor for the bacteriocin and the pore results from the peptide insertion and its oligomeric assembly in the lipid bilayer. To reveal which model is more suitable, we designed chimeric peptides fusing the membrane protein EtpM with different class II bacteriocins. Escherichia coli strains lacking the specific receptors for the bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they were called ?suicide probes?. Our results suggest that bacteriocins are potentially toxic as long as they are anchored to the membrane, either through their natural receptor, or through an artificial anchor such as EtpM. Hence, the receptor is more likely to act as a docking molecule bringing the bacteriocin closer to the membrane and it would be dispensable for the pore structure. Suicide probes allow to study in vivo interactions of bacteriocins with real bacterial membranes, complementing in vitro studies in model membranes. The effects of suicide probes in membrane fluidity and transmembrane potential were assessed using fluorescence spectroscopy. The data show that suicide probes increase phospholipid order and depolarize the membranes of receptor-free bacterial cells. We propose the use of suicide probes as a useful tool to gain more information about toxicity processes of bacteriocins in living cells, which is crucial to promote technological development of new antimicrobial peptides.