ENTEROCIN CRL35-­‐DERIVED SHORTER PEPTIDE PARTIALLY RETAINS ANTIMICROBIAL ACTIVITY

MASIAS E; SAAVEDRA L; BELLOMIO A; CILLI E; MINAHK C

San Miguel de Tucumán

Simposio; IV International Symposium on Lactic Acid Bacteria; 2013

Centro de Referencia para Lactobacilos

Enterocin CRL35 is a pediocin‐like bacteriocin that has been characterized in our group that proved to be effective in controlling the growth of the food‐borne pathogen Listeria monocytogenes. It is naturally produced by Enterococcus mundtii CRL35, a strain isolated from an Argentinean artisanal cheese (Tafi del Valle, Tucumán), but it is also available as a synthetic peptide for biochemical and biophysical studies. In this regard, 15‐mer peptides derived from native enterocin CRL35 were previously designed (peptides corresponding to residues 1‐15, 15‐29 and 29‐43). The N‐terminal‐ derived fragment was able to enhance the activity of enterocin CRL35 but 15‐mer peptides displayed no antimicrobial activity by themselves. Moreover, no antimicrobial activity was restored when all short peptides were combined. It was hypothesized that 15‐mer peptides were not long enough to span the plasma membrane in order to form a pore. Therefore, the objective of the present work was to continue this line of research by studying combination of the enhancer 1‐15 fragment with a newly designed 28‐mer peptide corresponding to residues 16‐43 as well as the complete synthetic enterocin CRL35. All peptide syntheses were manually performed using a solid‐ phase peptide synthesis protocol. Purity and identity were checked by mass spectrometry analyses. Strikingly, 28‐mer fragment alone was active against the sensitive strains L. monocytogenes FBUNT and L. innocua 7, although at higher concentrations as compared to native enterocin CRL35. Combinations with the other peptides were also studied in detailed by spot‐on‐lawn assays in agar plates as well as in liquid culture media where minimal inhibitory concentrations were calculated. Peptide binding to target cells was confirmed by fluorescence microscopy using fluorescent‐ derivatized peptides. To our knowledge, this is the first report showing residual anti‐Listeria activity of pediocin‐like bacteriocin fragments and it will contribute to the understanding of the mechanism of action of this class of antimicrobial peptides.