Covalent Structure, Synthesis, and Structure-Function Studies of Mesentericin Y 10537, a Defensive Peptide from Gram-positive Bacteria

Abstract

A 37-residue cationic antimicrobial peptide named mesentericin Y 105(37) was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 105(37) contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 105(37) was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 105(37) is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 105(37) in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30-40% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17-31. To reveal the molecular basis of the specificity of mesentericin Y 105(37) targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum alpha-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14-18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 105(37) are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative alpha-helical domain 17-31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1-14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 105(37) is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation.

Effect of Amino Acid Substitutions on the Activity of Carnobacteriocin B2

“…2). Recent studies on synthetic analogs of mesentericin Y105 support the hypothesis that highly conserved residues 1-14 of this 37-amino acid bacteriocin form part of a recognition sequence for a membrane-bound receptor but also indicate that the entire structure, including the N and C termini, plays a critical role in antimicrobial activity (14). Removal of Trp 37 or the N-terminal residues 1-3 from mesentericin reduces the inhibitory activity of mesentericin Y105 by a factor of 10 4 or more.…”

“…addition of a methyl group at the terminus of the valine residue), it is not surprising that the CbnI34 and CbnI37 bacteriocin mutants are fully active. The conservative differences between the YGNGV bacteriocins leucocin A (21,22) and mesentericin Y105 (17,18), namely replacement of Phe 22 by Ala and substitution of Val 26 by Ile in a 37-amino acid peptide, result in only slight variation in the levels of activity against identical organisms (14). The much more drastic alteration of Arg 46 in CbnB2 to Gly in CbnG46 did not affect the antimicrobial activity of the peptide, and this indicates that the side chain of this residue is not critical.…”

“…The insights gained into the structural requirements governing specificity of action, potency, and role as an inducer of bacteriocin production could serve as a guide to functional requirements for a host of closely related bacteriocins. A recent report on synthetic versions of mesentericin Y105, a 37-amino acid YGNGV bacteriocin, demonstrates that this peptide cannot be truncated at either the C or N terminus without loss of activity (14). In this study, we describe the successful overexpression, production, and purification from Escherichia coli of CbnB2, its precursor precarnobacteriocin B2, and six CbnB2 variants.…”

Selection and fitness in bacteriocin–producing bacteria

“…This variation is even lower if the bacteriocins of the genus Leuconostoc are surveyed. The leucocin group of bacteriocins produced by this genus shows very high homology at both the DNA and the amino acid level, and may differ in only one or two amino acids in the bacteriocin molecule between different strains or species (Hastings et al 1991Felix et al 1994 ;Fleury et al 1996). The high degree of similarity between the bacteriocins of the lactic acid bacteria shown across a number of phylogenetically distinct species, and even genera, is further highlighted by other features within this group.…”

“…Bacteriocins may act on cells in a variety of different ways (Reeves 1972 ;Jack et al 1995 ;James et al 1996). For example, many bacteriocins, such as mesentericin Y105$( and the B-colicins are membraneactive peptides which act to form pores in the cell membrane of antagonized cells (Fleury et al 1996 ;James et al 1996). These compounds cause leakage of ions and other cellular components, and in so doing disrupt the proton motive force, ultimately resulting in cell death (Abee et al 1994 ;Jack et al 1995).…”