Use of suicide probes to study class II bacteriocins mechanism of action against a receptor-free host

RIOS COLOMBO, N.S.; CHALON M.C.; BELLOMÍO A

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

The increasing resistance to common antibiotics is highly worrisome, thus, it has led to the study of alternative antimicrobials as a primary concern. Bacteriocins are a promising solution to this problem and the understanding of their mechanism of action is fundamental for development of new variants with potential use in the pharmaceutical industry. Class II bacteriocins are unmodified membrane-active 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. In order to reveal whether or not the pore structure involves a specific receptor, we designed chimeric peptides fusing the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35 (Ent35), pediocin PA-1 (PedA-1) and microcin V (MccV). We chose E. coli as an expression host because this bacterium is naturally insensitive to Ent35 and PedA-1, 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. If the fusion EtpM-bacteriocin kills the expressing host cell, it would mean that the specific receptor could be dispensable for the final step of membrane disruption. As these constructs exert a lethal effect when they are expressed, they are called ?suicide probes?. These hybrid proteins EtpM-bacteriocins were heterologeously expressed in E. coli and E. coli ΔsdaC respectively. The results suggest that, indeed, the specific receptor would act simply as a docking molecule and it would not participate in pore structure after the bacteriocin is anchored in the membrane. The effect of these suicide probes in some membrane properties is also analyzed.