RETHINKING THE MECHANISM OF ACTION OF CLASS II BACTERIOCINS: A COMPARATIVE STUDY THROUGH THE USE OF SUICIDE PROBES.

RIOS COLOMBO, N.S.; CHALON M.C.; DUPUY, F.G.; BELLOMIO, A.

Salta

Congreso; LV Reunion anual de la Sociedad Argentina de Investigaciones Bioquimica y Biologia Molecular; 2019

Sociedad Argentina de Investigaciones Bioquimica y Biologia Molecular

Class II bacteriocins are membrane-active peptides that act over a narrow spectrum of target bacteria. It is known that the antimicrobial activity of these compounds is carried out via pore formation in the membrane after recognition of a specific receptor. Nonetheless, little is known about the molecular events after the bacteriocin-receptor interaction. It is not clear yet whether the receptor is involved in the pore structure or if it only acts as a docking molecule for the bacteriocins. In this work, we grasped the previous design of ?suicide probes? that were first designed to study the insertion and topology of membrane proteins. We repurposed this tool to investigate the mode of action of different class II bacteriocins, namely microcin V (active against Gram-negative bacteria), pediocin PA-1, and enterocin CRL35 (active against Gram-positive bacteria). Suicide probes are hybrid peptides containing the different bacteriocins fused to the membrane protein EtpM. Arabinose-induced expression of these fusions attaches the bacteriocins to the periplasmic side of the Escherichia coli inner membrane. The constructs showed to be toxic for E. coli MC4100 cells lacking the specific receptor, thus supporting the role of the receptor as a binding site for bacteriocin anchoring to the membrane and not as a structural piece of the pore. In addition, microcin-based suicide probes are much more toxic than enterocin and pediocin-based fusions. Therefore, we discuss how the membrane composition, different in Gram-positive and Gram-negative bacteria, might be significant for bacteriocins activity. The developed probes were also applied to investigate and compare the effects of bacteriocins on membrane fluidity and transmembrane potential using fluorescence spectroscopy. The membrane-attached bacteriocins were proven to increase phospholipid order and depolarize the membranes of the receptor-free bacterial cells, upon arabinose induction. Since this system allows us to evaluate the interaction of bacteriocins in vivo with real bacterial membranes, we consider that it could be exploited to complement in vitro studies performed in model membranes, gaining molecular information of these and other antimicrobial peptides.