Advances in Characterizing the Transport Systems of and Resistance to EntDD14, A Leaderless Two-Peptide Bacteriocin with Potent Inhibitory Activity

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by the Enterococcus faecalis 14 strain previously isolated from meconium. This bacteriocin is mainly active against Gram-positive bacteria. Leaderless bacteriocins do not undergo post-translational modifications and are therefore immediately active after their synthesis. As a result, the cells that produce such bacteriocins have developed means of protection against them which often involve transport systems. In this and our previous work, we constructed different mutants deleted in the genes involved in the transport functions, thus covering all the supposed components of this transport system, using Listeria innocua ATCC 33090 as the indicator strain to assess the activity of externalized EntDD14. We also assessed the self-resistance of the WT and all its engineered derivative mutants against EntDD14, provided extracellularly, in order to evaluate their self-resistance. The results obtained highlight that the ABC transporter constituted by the DdG, H, I, and J proteins contributes to EntDD14 export as well as resistance to an external supply of EntDD14. Our results also have established the essential role of the DdE and DdF proteins as primary transporters dedicated to the externalization of EntDD14. Moreover, the in silico data showed that DdE and DdF appear to assemble in a formation that forms an essential channel for the exit of EntDD14. This channel DdEF may interact with the ABC transporter DdGHIJ in order to control the flow of bacteriocin across the membrane, although the nature of this interaction remains to be elucidated.