Suicide probes as tools to assess bacteriocin-membrane interactions in living bacterial cells

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

Montevideo

Encuentro; 4 th Protein Biophysics at the end of the world; 2019

Instituto Pasteur de Montevideo

The increasing resistance to common antibiotics is highly worrisome, thus, the study of alternative antimicrobials is a primary concern. Bacteriocins are a promising solution to this problem because they are membrane-active peptides that act forming pores over a narrow spectrum of bacterial targets. So far, there has been several studies on bacteriocin-membrane interactions mainly using model membranes. Although in vitro approaches are very useful to understand bacteriocins mechanism of action, these models demand to simplify an extremely complex and dynamic system such as the biological membrane. In this work we designed hybrid proteins called ?suicide probes? aimed to be expressed in bacterial hosts such as E. coli. This represent an in vivo system that allows to evaluate interactions of bacteriocins with real bacterial membranes, as a resource to complement studies in model membranes. The present work focus on how bacteriocin insertion affects bacterial membrane properties like fluidity and transmembrane potential, through the use of polarity-sensitive and potential-sensitive fluorophores (Laurdan and DisC3[5] respectively). The results demonstrate that the expression of the suicide probes tend to decrease membrane fluidity while producing a depolarization of the membrane. The arrangement of the lipids towards the gel phase make complete sense if we consider that a pore structure is being formed, where conformational changes are likely to happen in both the peptides and the phospholipids. The simultaneous dissipation of the transmembrane potential and the deleterious effect on the cell are in line with the processes associated with the pore formation, where an efflux of ions and essential molecules is taken place. The suicide probe system is a good start to gain more information about toxicity processes of bacteriocins in living cells. We propose this approach as a platform to assess several peptide and membrane properties in physiological conditions.