Suicide probes and Laurdan as tools to assess in vivo bacteriocinmembrane interactions

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

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

Class II bacteriocins are membrane-active peptides that act forming pores over specific target bacteria. Their study is a primary concern, since they are considered alternative antimicrobials. 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 is an in vivo system that allows to evaluate interactions of bacteriocins with real bacterial membranes, as a tool to complement studies in model membranes. On the other hand, Laurdan is a popular fluorophore, deemed to work extremely well not only in model membranes but also in living cells. It is used to assess changes in membrane fluidity that result from phase transitions. The present work provides a dataset of fluorescence emission spectra and GeneralizedPolarization (GP) values of Laurdan-labeled E. coli bacterial cells, expressing three engineered class II bacteriocins. The results demonstrate that the insertion of different bacteriocins tend to increase membrane order. 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. Additionally, control experiments were performed to evaluate how accurate is Laurdan as an indicator of E. coli membrane fluidity changes. It is well known that bacterial cells rapidly adapt to changes in temperature by adjusting the fatty acid composition of the membrane, in order to maintain the homeostatic parameters. Thus, fluorescence spectra of Laurdan-labeled E. coli cells are compared, to check the GP behavior in response to different growing temperatures.