Suicide probes and Laurdan as tools to assess in vivo bacteriocin membrane interactions

RÍOS COLOMBO NS; CHALÓN MC; DUPUY FG; BELLOMIO A

San Luis

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

SAB

Class II bacteriocins are membrane-active peptides that act forming pores over specifictarget bacteria. Their study is a primary concern, since they are considered alternativeantimicrobials. So far, there has been several studies on bacteriocin-membraneinteractions, mainly using model membranes. Although in vitro approaches are veryuseful to understand bacteriocins mechanism of action, these models demand to simplifyan extremely complex and dynamic system such as the biological membrane. In thiswork we designed hybrid proteins called ?suicide probes? aimed to be expressed inbacterial hosts such as E. coli. This is an in vivo system that allows to evaluateinteractions of bacteriocins with real bacterial membranes, as a tool to complementstudies 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 isused to assess changes in membrane fluidity that result from phase transitions. Thepresent work provides a dataset of fluorescence emission spectra and GeneralizedPolarization (GP) values of Laurdan-labeled E. coli bacterial cells, expressing threeengineered class II bacteriocins. The results demonstrate that the insertion of differentbacteriocins tend to increase membrane order. The arrangement of the lipids towardsthe 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 thephospholipids. Additionally, control experiments were performed to evaluate howaccurate is Laurdan as an indicator of E. coli membrane fluidity changes. It is well knownthat bacterial cells rapidly adapt to changes in temperature by adjusting the fatty acidcomposition of the membrane, in order to maintain the homeostatic parameters. Thus,fluorescence spectra of Laurdan-labeled E. coli cells are compared, to check the GPbehavior in response to different growing temperatures.