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

Graphical abstract Highlights ► A chimerical gene encoding a hybrid bacteriocin–microcin was generated by PCR. ► Ent35–MccV is a hybrid bacteriocin containing enterocin CRL35 and microcin V. ► Ent35–MccV is active against Gram-positive and Gram-negative bacteria. ► Enterohemorrhagic Escherichia coli and Listeriamonocytogenes are sensitive to Ent35–MccV.


Introduction
Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides [1]. Many food pathogens as well as opportunistic bacteria that cause food spoilage are inhibited by these peptides. Particularly, bacteriocins from lactic acid bacteria (LAB) are being employed as antimicrobial agents for foods. In this regard, nisin and pediocin PA-1 were approved as food biopreservatives [2]. Actually, nisin was approved as food antimicrobial agent in 1969 by FAO/WHO Expert Committee on Food Additives and its use is permitted in over 50 countries nowadays. Biopreservation methods are being developed to respond to the market demand of fresh and minimally processed foods with longer shelf-life. One of the critical limitations to the use of LAB bacteriocins as biopreservative agents is the narrow spectrum of action they display. As a matter of fact, pediocin PA-1 is inactive against Gram-negative bacteria and nisin has to be employed in combination with chemical or physical treatments that disrupt the outer membrane (OM) when used to inhibit Gram-negative pathogens [3,4]. Pathogenic strains of Escherichia coli can cause different foodborne illnesses. The most common of them is gastroenteritis. Moreover, Shiga toxinproducing E. coli is a foodborne pathogen causing alarming outbreaks. A possible alternative to control Gram-negative in foods is the use of antimicrobial peptides secreted by Gram-negative bacteria, known as microcins. To this purpose, Pomares et al. developed a chymotrypsin-susceptible MccJ25 variant, inactivated by digestive enzymes to be used as food preservative [5]. However, microcins are only active against Gram-negative bacteria [6]. Similar to pediocin-like bacteriocins, microcins belonging to class IIa, such as microcin V, are linear polypeptides and the removal of the leader peptide is the unique post-translational modification that they undergo before being secreted by the cells [6][7][8]. Most remarkable is the fact that they have a similar mechanism of action. It was found that many linear bacteriocins can disrupt the cellular membrane and kill sensitive microorganisms. The expression of an integral membrane protein that acts as a bacteriocinspecific receptor determines the sensitivity of a bacterial strain to the bacteriocin. The mannose phosphotransferase system acts as receptor for pediocin-like bacteriocins [9]. Meanwhile, three different proteins may serve as a specific receptor for linear microcins, namely the membrane component F 0 of the ATP synthase, SdaC, and the mannose permease, required by MccH47, MccV, and MccE492, respectively [10][11][12]. Because of the Gramnegative envelope structure, an additional step is required by class IIa microcins, i.e. an OM transporter system is used for these peptides to reach the plasma membrane receptor.
The enterocin CRL35, a pediocin-like bacteriocin isolated from Argentinean regional cheese, has a potent antilisterial activity but is inactive against Gram-negative bacteria [13]. On the other hand, microcin V previously known as colicin V is specifically active against Gram-negative bacteria [14]. In order to obtain a peptide with a broader antimicrobial spectrum the required portions of genes encoding the bacteriocins enterocin CRL35 and microcin V, namely munA and cvaC, were fused by asymmetrical PCR. The resulting munA-cvaC chimerical gene was cloned and expressed in E. coli. The hybrid bacteriocin purified from E. coli extracts, named Ent35-MccV, showed inhibitory activity against enterohemorrhagic E. coli, Listeria monocytogenes, and other pathogenic Gram-positive and Gram-negative bacteria.

Bacterial strains and culture conditions
The bacteria and culture conditions used in this work are listed in Table 1. The strain E. coli DH5a was used as the host for recombinant plasmids.

Genetic manipulations
In this work we fused by polymerase chain reactions (PCR) the regions of munA and cvaC encoding for the structural portion of enterocin CRL35 and microcin V, respectively. The procedure is shown schematically in Fig. 1A. A colony of Enterococcus mundtii CRL35 as template and the primers munAF3 (forward-5 0 CAT GCCATGGGTAAATACTACGGTAATGGA) and 3GentR (reverse-5 0 TC CTCCTCCACTTTTCCAACCAGCTGCTC) [13] were used for amplifying munA gene. On the other hand, the gene cvaC was amplified from the plasmid pHK11 using primers colVF3G (forward-5 0 GGAG GAG GACTTCAGGGCGTGATATTGC) and colVR (reverse-5 0 GGATCCTTA TAAACAAACATCACT) [15]. Recognition sites for restriction enzymes NcoI in munAF3 and BamHI in colVR and the region encoding for the hinge triglycine in 3GentR and colVF3G are underlined. The reaction was carried out under standard conditions specified by the enzyme supplier (AccuPrime™ Taq DNA Polymerase High Fidelity, Invitrogen).The PCR program included an initial denaturation step at 94°C for 5 min, followed by 30 cycles (50°C for 30 s, 68°C for 40 s and 94°C for 1 min). At the end of the PCR program, an elongation step (68°C, 5 min) was included.
Finally, the fusion of munA with cvaC was carried out by an asymmetric PCR reaction [16]. The PCR products of munA and cvaC (1 ll each one in a 50 ll reaction) were used as template, munAF3 and colVr as primers at 0.4 lM final concentration and the internal munAR3g primer at 0.4 nM final concentration was introduced to produce the fusion. Nucleotide concentrations and reaction conditions were those recommended by the supplier of the enzyme GoTaq DNA Polymerase (Promega). The program used was: 94°C 5 min, followed by 10 cycles (94°C 1 min, 55°C 1 min, 72°C 40 s), 20 cycles (94°C 1 min, 40°C 1 min, 72°C 40 s) and 72°C 10 min. The PCR product (shown in Fig. 1B) was purified from  agarose gel using a Gel Band Purification Kit (GE Healthcare), digested with NcoI and BamHI and cloned in linearized pET28b(+) (Novagen). The ligation mixture was used to transform E. coli DH5a obtaining pMA24. The cloned gene was sequenced and analyzed for its structure. E. coli LA1 and LA2 were obtained introducing a kanamycin resistance cassette by Phage P1 vir transduction from the strains E. coli JW2767-1 and JW2142-1, respectively [17]. The strain E. coli NC1 was a generous gift from Natalia Corbalán (INSIBIO, Tucumán-Argentina).

Gene expression and bacteriocins purification for activity determinations
Ent35-MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37°C. When the culture reached an OD $0.6 the Ent35-MccV synthesis was induced with isopropyl b-thiogalactoside 0.5 mM and temperature was changed to 30°C.

RP-HPLC purification of Ent35-MccV and MALDI-TOF MS analysis
The supernatant obtained from the centrifugation of the heated extract (Section 2.3) was precipitated with ammonium sulfate to a final concentration of 70% (w/v). Proteins were harvested by

Antimicrobial activity and bacterial viability assays
Drops of each purified peptide (20 ll, 350 lg of protein/ml) were spotted in a TSB (Difco) or M9 (Sigma) plate. Once the droplets were adsorbed, the plate was covered with 4 ml of 0.6% agar previously inoculated with 10 6 cells of the test strain. Incubation was carried out over night at 30°C or 37°C. Antimicrobial activity was evaluated by measuring the diameter of the inhibition halos. The peptide solutions were successively diluted before the drops were spotted. The antimicrobial activity units (AU/ml) were calculated as the reciprocal of the highest two-fold dilution showing growth inhibition halo against Listeria innocua 7 or E. coli MC4100.
For viability determination E. coli MC4100 and L. innocua 7 were grown to mid-log phase, harvested by centrifugation (10 min, 10000xg), and re-suspended at approximately 1.10 7 colony forming units per milliliter (CFU/ml) in 50 mM HEPES, pH 7.4, 12.5 mM glucose. The suspensions were treated with the bacteriocin (50 AU/ml corresponding to each assayed bacterial strain) and the CFUs versus time were determined.

Tricine SDS-PAGE
Electrophoresis gels were prepared as previously described [19] and the samples were treated with loading buffer without b-mercaptoethanol. After fixing, the gels were washed successively with ethanol 70%, 30% and sterile bidistilled water. The gels were placed on TSB or LB (Sigma) agar plates, covered with 10 ml of 0.6% (w/v) agar inoculated with 10 6 cells of L. monocytogenes FBUNT1 or E. coli MC4100. The plates were incubated at 37°C for 16 h and the inhibition zones were examined.

Nucleotide sequence accession number
The DNA sequence of munA-cvaC was deposited at the EMBL Nucleotide Sequence Database, EMBL-Bank ID: FR865441.

Construction of munA-cvaC
As described in Section 2.2, the hybrid munA-cvaC was obtained by asymmetrical PCR (see Fig. 1A). First, each gene was amplified using the previously described primers. The DNA sequence encoding the hinge region was incorporated with 3GentR (reverse primer for munA) and colVF3G (forward primer for cvaC) and overlapping fragments were generated. After that, the two fragments were fused by an asymmetrical PCR (Fig. 1A) [20]. The hybrid gene was cloned, expressed in E. coli BL21 [DE3] and purified by reverse phase chromatography. The hinge region between two antimicrobial peptides was reported to be critical for obtaining active antimicrobial hybrid peptides [21]; thus, a Gly-Gly-Gly flexible region was introduced between enterocin CRL35 and MccV. The amino acid sequence of parental and the hybrid Ent35-MccV are shown in Fig. 1C.

Spectrum of action
As shown in Table 1, the antimicrobial spectrum of Ent35-MccV was assayed against a number of Gram-positive and Gramnegative bacteria. In agreement with well-established data, none of the Gram-positive strains tested were inhibited by MccV and enterocin CRL35 was not active against Gram-negative bacteria. Similar to the parental bacteriocins, the hybrid peptide displayed potent antimicrobial activity against Listeria spp. and E. coli. Ent35-MccV was active against L. innocua 7, two clinical isolated strains of L. monocytogenes, namely FBUNT1 and FBUNT2, and was inactive against the enterocin CRL35 natural resistant strain L. monocytogenes EGDe. All the Gram-negative bacteria inhibited by MccV were also inhibited by Ent35-MccV. In particular, the peptide displayed antimicrobial activity against Enterobacter cloacae and E. coli; including the laboratory strain MC4100, the enterohemorrhagic E. coli O157:H7 and 5 out of 6 clinical isolates of uropathogenic strains (UPEC). In fact, only the strain UPEC230 was not affected by Ent35-MccV. However, UPEC230 is a microcinogenic strain that produces an antimicrobial compound which was characterized by cross-streaking as MccV [22]. It was previously suggested that a relationship between higher-molecular-mass microcins, such as MccV and urovirulence may occur [23]. Moreover, Ent35-MccV displayed antimicrobial activity against other clinical isolates Gram-positive and Gram-negative strains on which enterocin CRL35 or MccV are inactive, such as Staphylococcus aureus FBUNT1 and FBUNT2, Staphylococcus epidermidis, Serratia marscecens FBUNT1 and Klebsiella pneumoniae FBUNT1. The hybrid peptide was inactive against Enterococcus faecalis FBUNT1, on which enterocin CRL35 is active. Some bacteria tested (Acinetobacter baumannii, Yersinia pestis, Citrobacter freundii, Pseudomonas aeruginosa, Salmonella enterica serovar Typhimurium, and S. enterica serovar Newport) were resistant.

Mechanism of action
To explore the mechanism of action of Ent35-MccV, its antimicrobial activity was assayed against wild type E. coli MC4100 and isogenic mutant strains, bearing mutations in the cirA, tonB or sdaC genes. All the mutant strains are resistant to MccV because CirA and TonB are necessary to transport the peptide across the OM [24], and SdaC is the IM receptor [11]. The results showed that the mutant strains (E. coli LA1, LA2 and NC1) were also resistant to Ent35-MccV, indicating that the same proteins are required by the hybrid bacteriocin in order to exert antimicrobial action on E. coli.
Both, Enterocin CRL35 and MccV are bactericidal [11,25]. To investigate whether Ent35-MccV is able to kill the target cells, we studied the time course killing effect on E. coli MC4100 and L. innocua 7 using the same AU/ml (Fig. 2). Ent35-MccV was able to decrease the viability of both bacteria. After two hours of treatment with Ent35-MccV the L. innocua 7 viable cells decreased one order of magnitude compared to the control curve in the absence of the peptide. A similar result was obtained with enterocin CRL35 (Fig. 2A). When the experiments were carried out with E. coli as sensitive strain, cell viability decreased two orders of magnitude by treatment with Ent35-MccV, meanwhile the same antimicrobial activity units of MccV decreased only one log, compared to the control without antimicrobial peptide (Fig. 2B). These results clearly demonstrate the bactericidal ability of Ent35-MccV against both bacteria.

Ent35-MccV characterization
The MALDI-TOF MS analysis of Ent35-MccV samples prepared as described in Sections 2.3 and 2.4 showed a single signal at m/z 7271.6. According to previously published MS data [26,27] this component was tentatively identified as to the E. coli coldshock-C secreted protein.
The missed detection of any further protein component has to be most likely ascribed to a high ionization efficiency of the CSP-C and to subsequent suppression ion effects. Therefore, the sample was subjected to preliminary RP-HPLC separation. In Fig. 3A and B are shown the UV chromatograms at k = 220 and k = 280, respectively. Peaks were manually collected and analyzed by MALDI-TOF MS. The dominant component with m/z 7271.6 was contained in the peak no. 1 at retention time (RT) 36.7 min. The MALDI-TOF peptide mass mapping carried out after tryptic hydrolysis (not shown) confirmed, through the use of the Protein Prospector search engine (http://prospector.ucsf.edu), that it is the CSP-C of E. coli (Uniprot accession: P0A9Y6). Though at a low degree, the CSP-C also contaminated the later eluting HPLC fractions.
The MALDI-TOF spectrum of the peak no. 5 at RT 46.2 min (Fig. 4) displayed a signal at m/z 13179.8. The measured MW is compatible with that expected for Ent35-MccV in which a proteolytic cleavage at the Gly 1 has occurred. Interestingly, the corresponding chromatographic peak resulted clearly increased by the k = 280 detection (Fig. 3B), if compared to the other peaks. This was attributed to the presence of as many as 4 Trp residues in the sequence of Ent35-MccV, that strongly enhanced k = 280 absorbance.
The remaining main signals in the MALDI spectrum can be assigned to other co-eluting fragments that arise from proteolysis of Ent35-MccV as schematized in Table 2, thereby supporting the identification of Ent35-MccV. The proteolytic cleavages preferentially involved the carboxy-terminal of specific Gly residues. No further polypeptide chains ascribable to fragments of Ent35-MccV were detected by the MS analysis of the remaining HPLC fractions. It has to be underlined that the attempt of characterizing Ent35-MccV by MALDI peptide mass mapping was unsuccessful, most likely because of both a low degree of trypsinolysis of the chimeric protein and the occurrence of interfering peptides generated from E. coli CSP-C.

Tris-tricine SDS-PAGE and Ent35-MccV assay
Purified preparations containing Ent35-MccV and the parental bacteriocins were analyzed by tricine SDS-PAGE. Gels revealed by  biological activity against L. monocytogenes FBUNT1 and E. coli MC4100 are shown in Fig. 5A and B, respectively. Ent35-MccV inhibition zones were located at $13.3 kDa (lane 2, band (a) for both gels), close to the migration band expected for unhydrolyzed Ent35-MccV (theoretical MW 13259.8 Da). These results demonstrated that the hybrid peptide is actually responsible for the antimicrobial activity, different from any small-sized proteolytic fragment produced during the purification. There is an additional inhibition zone (c), which could be attributed to Ent35-MccV oligomers. It was only observed when antimicrobial activity was tested against E. coli MC4100 SDS-PAGE detection (Fig. 5B).

Discussion
In this paper, a new hybrid bacteriocin between the pediocinlike bacteriocin, enterocin CRL35 and the linear microcin, MccV is reported. Johnsen et al. reported the fusion of N-and C-terminal portions of various ''pediocin-like'' bacteriocins and found that the C-terminal half of the peptide determines the spectrum of action against species and strains of Gram-positive bacteria [28,29]. Moreover, a 15-mer peptide that spans pediocin PA-1 from the center toward the C-terminal portion inhibited its biological activity. Thus, the receptor binding region could be localized within the C-terminal portion of these bacteriocins [30]. The N-terminal region must be involved in the membrane perturbation induced by the peptide, since a 15-mer peptide derived from enterocin CRL35 produced an enhancement of enterocin CRL35 antimicrobial activity and showed antimicrobial activity [31,32]. On the other hand, Azpiroz and Laviña constructed hybrid microcins fusing the N-and C-terminal domains of MccV and MccH47. They arrived to the conclusion that the C-terminal domains of these class II microcins are involved in the recognition of the OM receptor for the target cell uptake and the N-terminal domain is the toxic region of the molecule [33]. Both, class IIa of LAB bacteriocin and class II of microcins must have an IM receptor binding region.
The antimicrobial spectrum displayed by Ent35-MccV demonstrates that both bacteriocins are active in the chimerical peptide. Ent35-MccV must have two receptor binding regions, two toxic regions and the OM receptor binding domain in the MccV portion. Although the hybrid bacteriocin appears to have a similar mechanism of action than the parental bacteriocins and is recognized by the two immunity proteins, i.e. MunC and Cvi, the antimicrobial activity of the hybrid does not correspond exactly to the sum of the antimicrobial activities of the parental peptides. The toxic domains belonging to both peptides might be active simultaneously once Ent35-MccV binds to the plasma membrane receptor in E. coli cells. This fact could explain the better capacity to kill E. coli cells than MccV and the better performance killing E. coli than L. innocua cells. There is a persistence of L. innocua 7 and E. coli MC4100 populations larger than 1.10 6 CFU/ml and 1.10 5 CFU/ml after treatment with the hybrid antimicrobial peptide. Vignolo et al. observed that population of L. monocytogenes decreased initially after enterocin CRL35 treatment and increased upon further incubation [34]. Another microcins and bacteriocins have similar kinetics of cell destruction [35,36]. Although more experiments testing the antimicrobial activity of Ent35-MccV in foods or complex fluid matrices (whole blood, plasma or serum) must be performed, in order to find the right conditions in which the spontaneous emergence and outgrowth of a bacteriocinresistant bacterial population can be avoided. In fact, we strongly believe that Ent35-MccV could be employed in combination with other bacteriocins or other chemical or physical food treatments [4,34,37].
The antimicrobial activity displayed by the hybrid against bacterial strains which are not inhibited by the parental bacteriocins  could be explained by the presence of multiple domains or modules in Ent35-MccV. It is possible that a membrane protein of the target cells serves as a receptor for one of the parental bacteriocins or Ent35-MccV. However, a steric barrier or an inappropriate bacteriocin position could impede the interaction between the toxic domain and the cellular membrane. Therefore, the parental bacteriocins could not act causing the disruption of the cell membrane. After receptor binding, the hybrid peptide could have increased possibilities for a favorable toxic domain-membrane interaction. These results must be thoroughly investigated in future research in order to gain more insight into the mechanism of action of membrane active bacteriocins. Ent35-MccV is active against the emergent foodborne pathogens, the Gram-negative enterohemorrhagic E. coli O157:H7 and the Gram-positive L. monocytogenes. More broad spectra hybrid bacteriocins could be obtained based on the idea proposed in this work. Furthermore, chimerical bacteriocin-microcin peptides must be useful to study the mechanism of action of bacteriocins and for applications in medicine, veterinary or food industry.