A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria

Abstract

Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35-MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35-MccV may find important applications in food or pharmaceutical industries.

Keywords: AU, antimicrobial activity units; Bacteriocin; CFU, colony forming units; Enterocin CRL35; Hybrid antimicrobial peptide; IM, inner membrane; LAB, lactic acid bacteria; Mcc, microcin; Microcin; Microcin V; OM, outer membrane.

Graphical abstract

Fig. 1

munA-cvaC construction. (A) Schematic representation of the PCR strategy used to fuse munA and cvaC. First, each gene was amplified incorporating an overlapping fragment which encode for an interpeptide flexible region. In the second PCR, the products of munA and cvaC PCR were used as template and 3GentR (1:1000 dilution with respect to munAF3 and colVR primers) was used to fuse the two DNAs. (B) Electrophoretic analysis of the product of the asymmetrical PCR in 1% agarose gel stained with (Gelred^TM (Biotium). Lane 1: DNA step ladder 100 bp (Invitrogen). Lane 2: asymmetric PCR product. The arrow marks the position of migration of the hybrid product. (C) Amino acid sequences of enterocin CRL35, MccV and the hybrid Ent35–MccV. The hinge region is in bold.

Fig. 2

Effect of Ent35–MccV and parental bacteriocins on the viability of L. innocua 7 and E. coli MC4100 cells. The bacterial suspensions were treated with 50 AU/ml of each bacteriocin (see Section 2.4). Cell viability is expressed as CFU/ml at different times. (A) L. innocua 7 cells without treatment (○), Ent35–MccV (■), and enterocin CRL35 (Δ). (B) E. coli MC4100 control without treatment (○), Ent35–MccV (■), and MccV (Δ). The results represent the average of three independent experiments.

Fig. 3

RP-HPLC separation of the heated cellular extract. Cellular extract of E. coli BL21 [DE3] (pMA24) were treated as described in Section 2.4. Chromatogram was monitored at (A) λ = 220 nm and (B) λ = 280 nm. Peaks were collected and individually analyzed by MALDI-TOF MS.

Fig. 4

MALDI-TOF MS analysis of the HPLC fraction no. 5. The mass signal 13179.8 is compatible with the expected molecular weight of Ent35–MccV (2–135 chain). The other signals are assigned in Table 2. The upper panel shows the Ent35–MccV sequence in which the proteolytic cleavage sites are indicated.

Fig. 5

Bacteriocins analyzed by Tris-Tricine SDS-PAGE and revealed by biological activity. Lane 1 contained the prestained molecular mass marker – see Blue Plus2, Invitrogen (both gels). (A) Gel revealed by biological activity against L. monocytogenes FBUNT1 showing the zones of growth inhibition by the bacteriocin bands (a) and (b). Lanes: 2, Ent35–MccV; 3, enterocin CRL35 (MM: 4289.8 Da). (B) Gel revealed using E. coli MC4100 as indicator strain. Lanes: 2, Ent35–MccV (band a); 3, MccV, MM: 8735.7 Da (band b).