A synthetic analog of plantaricin 149 inhibiting food‐borne pathogenic bacteria:evidence for α‐helical conformation involved in bacteria–membrane interaction

Abstract

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277-282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane. Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014. The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an alpha-helix. In this membrane model, the hydrophobic side of the alpha-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface.

Contribution of the Tyr-1 in Plantaricin149a to Disrupt Phospholipid Model Membranes

“…Similar behavior was observed to the peptide’s antimicrobial activity against P. aeruginosa , which presented the same MIC value of 310 μM for Pln149a and Pln149S (369 and 363 μg/mL, respectively), and a more elevated MBC to Pln149S (310 μM) than to Pln149a (155 μM). Similar to PlnA, Pln149a could be considered a weak antimicrobial agent due to the high MIC values found here, and as already described of this peptide [28,29,31]. Nevertheless, the motivations for studying the mechanism of action of this antimicrobial peptide against different classes of microorganisms are: its stability in acid pH ranges, its inactivation by humans digestive proteases (which is of particular interest for suggesting its application as a nontoxic and safe food preservative to control the microflora of fermented foods, with no effect against the microflora of the human intestine).…”

“…The first analog was Pln149a, the C -terminus amidated version of the natural Pln149 antimicrobial peptide, as previously described [28,29]. The second analog was Pln149S, a peptide in which the Tyr residue at position 1 ( N -terminal end) was substituted by a Ser residue (Y1S substitution).…”

“…The antimicrobial activity of PlnA was shown to be unspecific [26] and with high MIC values reported, since the natural function of the peptide was suggested to act as a pheromone. In a similar way to PlnA, the peptide Plantaricin149 (Pln149), produced by L. plantarum NRIC 149, is also cationic in nature, composed of 22 amino acid residues with inhibitory activity against some pathogenic bacteria [27,28] with high minimum inhibitory concentration (MIC) values [27,29], compared to the MIC of other LAB bacteriocins [26]. The mode of action proposed to the amidated analog, Pln149a, (charge +7 at pH 7.0) assumes it adopts an unordered conformation in aqueous solution [28,29]; however, upon binding to negatively charged membranes, Pln149a changes to an α-helix conformation.…”

“…In a similar way to PlnA, the peptide Plantaricin149 (Pln149), produced by L. plantarum NRIC 149, is also cationic in nature, composed of 22 amino acid residues with inhibitory activity against some pathogenic bacteria [27,28] with high minimum inhibitory concentration (MIC) values [27,29], compared to the MIC of other LAB bacteriocins [26]. The mode of action proposed to the amidated analog, Pln149a, (charge +7 at pH 7.0) assumes it adopts an unordered conformation in aqueous solution [28,29]; however, upon binding to negatively charged membranes, Pln149a changes to an α-helix conformation. The molecular model of the whole Pln149a sequence, created by SP 3 software, is a helix structure which was proposed to extend from the residue Ala7 to Lys20 [28] in an amphipathic structure with the polar residues K11, K14, K15, K18, and K19 of Pln149a along one side of the helix and the nonpolar residues, I10, V13, L16, and F17 along the other.…”

“…The mode of action proposed to the amidated analog, Pln149a, (charge +7 at pH 7.0) assumes it adopts an unordered conformation in aqueous solution [28,29]; however, upon binding to negatively charged membranes, Pln149a changes to an α-helix conformation. The molecular model of the whole Pln149a sequence, created by SP 3 software, is a helix structure which was proposed to extend from the residue Ala7 to Lys20 [28] in an amphipathic structure with the polar residues K11, K14, K15, K18, and K19 of Pln149a along one side of the helix and the nonpolar residues, I10, V13, L16, and F17 along the other. A helical conformation was observed in the peptide’s structure when in the presence of sodium-bis-(2-ethylhexyl)-sulfosuccinate (AOT) micelles, trofluoroethanol (TFE) [29], and negatively charged vesicles [30].…”