Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

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Abstract

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure–activity relationship in the N-terminal domain of these peptides, three analogues (13) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.

Introduction

Bacteriocins are potent antimicrobial peptides with a potential in a variety of applications such as food preservation, replacement of conventional antibiotics, and treatment of bacterial multiple-drug-resistance.1, 2, 3 Bacteriocins derived from lactic acid bacteria are produced as a part of their self defense mechanism to destroy competing microorganisms.4 Bacteriocins like class I nisin and class IIa pediocin PA-1 are currently used for food preservation and their potential in medical applications is being explored.1 Class IIa bacteriocins are unmodified cationic peptides ranging from 37 to 48 residues that display activity against a variety of food pathogens, including Listeria monocytogenes in the nanomolar range.2

Bacteriocins display potent activity by one or more of the proposed mechanisms of actions. These peptides can (i) permeabilize the target cell membrane and reduce the proton motive force of bacterial cells, (ii) induce lysis of the cell by activating autolysins1, 5, 6 or (iii) bind to the membrane bound receptor and cause pores in the cell wall.7, 8 For class IIa bacteriocins, it is becoming clear that peptides act by interacting with a specific receptor, mannose phosphotransferase system (PTS) permease, on the target cell membrane and for this peptide–receptor interaction folded conformation of the peptide is a key element. Comparison of the amino acid sequence of class IIa bacteriocins shows that these peptides consist of a highly conserved hydrophilic and charged N-terminal region harboring the consensus YGNGV sequence and a disulfide bond. The C-terminal region is hydrophobic with a more variable sequence. In contrast, the three dimensional solution structures of several class IIa bacteriocins show that N-terminus region folds into different conformations, such as anti-parallel β-sheet or coiled structure, and the C-terminal region maintains a conserved amphipathic α-helical structure.9, 10, 11 The C-terminal helical region imparts target specificity and likely interacts with the target cell membrane bound receptor, whereas the positively charged N-terminal region interacts with the cell surface by electrostatic interaction.12, 13, 14

Leucocin A (LeuA) is a 37 residue class IIa bacteriocin from Leuconostoc gelidium.15 A series of experiments conducted by Fleury et al.16 to elucidate structure activity relationship (SAR) of LeuA show that N-terminal YGNGV sequence and C-terminal tryptophan are essential for activity. The N-terminal of LeuA (residues 2–16) consists of a three stranded anti-parallel β-sheet which is stabilized by the disulphide bond between residues 9 and 14, and C-terminal residues Trp18-Ala30 form an amphipathic α-helix. (Fig. 1a).9, 17 The conserved disulphide bond between two cysteines maintains the correct geometry of other residues in the sequence and this geometry is essential for its activity.18 Studies conducted by Derksen et al.18 confirmed that the disulphide bond only contributes to maintain correct geometry in the molecule but does not bind to the receptor on the bacterial cell membrane. The authors substituted the disulphide bond with carbocyclic rings without losing activity while maintaining flexibility and geometry in the synthetic LeuA. More recently it was shown that the replacement of the two Cys residues in the N-terminal region of LeuA with hydrophobic residues such as Phe, Nva (Norvaline), or AllylGly yields a fully active analogue.19 On the other hand, substitution of the Cys with Ser residues in LeuA made the peptide inactive. These results suggests that although the disulfide bond is conserved among class IIa bacteriocins, it can be replaced with residues which can induce similar geometry in the molecule without losing its antibacterial activity.

We hypothesized that analogues of LeuA can be designed by replacing a portion of the N-terminal β-sheet region (Cys9-Ser15) with a smaller β-turn. To validate this and study the SAR of LeuA, three analogues (13) of LeuA were designed and synthesized by manipulating the N-terminal region of the native LeuA (Fig. 1). LeuA was synthesized as a control employing native chemical ligation (NCL) of two smaller fragments of LeuA. The antimicrobial activity of analogues and LeuA was evaluated against two indicator strains, Carnobacterium divergens and Listeria monocytogenes. In general the analogues did not show any activity whereas synthetic LeuA was highly active. Interestingly, analogue 1 was able to competitively inhibit the activity of LeuA. Solution conformation study using circular dichroism (CD) spectroscopy suggested that only analogue 1 most likely adopts similar helical folding as LeuA. Further, molecular dynamics (MD) simulations of 1 revealed that the C-terminal region folds into a well-defined α-helix, however, the N-terminal region does not form a β-sheet structure like the native LeuA emphasizing the role of proper folding in the activity of class IIa bacteriocins.

Section snippets

Design of LeuA analogues

As mentioned above, the N-terminal region of LeuA mainly interacts with electrostatic interaction with the target cell membrane. Therefore, the exact role of folded conformation in the N-terminal region of class IIa bacteriocins is not clear. We designed three analogues of LeuA by replacing the N-terminal β-strand residues ranging from Cys9-Asn17 with smaller β-turn sequences (Fig. 1). The analogues were 32-residue long, five residues less compared to the native LeuA. Lys11 was kept in all the

Conclusion

In conclusion, LeuA analogues (13) studied here support earlier investigations that the C-terminal region is required for specificity and dictates the antimicrobial profile, whereas, the N-terminal sequence is necessary for activity. Replacement of few N-terminal residues with conservative substitutions is allowed as shown previously,19 however, the complete N-terminal domain is required for activity. Further, we found that NCL is a better approach for the solid phase synthesis of LeuA like

Synthesis of peptide analogues

Synthesis of peptide analogues 13 was carried out on 1.0% DVB cross-linked chlorotrityl resin (0.2 mmol, loading 1.05 mmol/g) following the standard Fmoc solid-phase peptide chemistry with acid labile side chain protections (t-Bu, Boc, Trt) as described previously.36 Peptides were synthesized using MPS 357 automated peptide synthesizer robot (Advanced Chemtech Inc., USA). To assist the coupling of difficult residues (Asn, Ser, and for all residues after position 10), increased coupling times

Acknowledgments

We thank Wael Soliman for assistance with MD simulations, Sahar Ahmed for help in writing, and Liru Wang (CanBiocin Inc.) for assistance in performing antimicrobial assays. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). The infrastructure support from the Canada Foundation for Innovation (CFI) is also acknowledged.

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