Towards a new approach for class II bacteriocins: using "suicide probes" to study their mechanism of action

RÍOS COLOMBO, N.S.; CHALÓN, M.C.; DUPUY, F.G.; BELLOMIO, A.

Lima

Congreso; Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level; 2019

Biophysical Society

The study of alternative antimicrobials has become relevant in the last years because of the increasing resistance to common antibiotics. Class II bacteriocins are unmodified membraneactive peptides that act over a narrow spectrum of bacterial targets. They are believed to bind a specific receptor on the membrane that would participate in the formation of a pore, leading to membrane permeabilization and cell death. Objectives: 1) Reveal whether or not the pore structure involves the specific receptor. 2) Study the effect three bacteriocins in some membrane properties using fluorescent spectroscopy. Methods: we designed chimeric peptides fusing the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and Microcin V. These hybrid proteins EtpM-bacteriocin (also called ?suicide probes?) were heterologously expressed in E. coli and E. coli ΔsdaC respectively. We chose E. coli as an expression host because this bacterium is naturally insensitive to enterocin and pediocin, since their specific receptor Man-PTS is not present on its inner membrane. In addition, an sdaC mutant E. coli strain was employed as a receptor-free host for MccV, as it does not express SdaC, the specific membrane receptor for this microcin. The effect of these suicide probes on transmembrane potential and membrane fluidity was assessed.