Engineering lantibiotic-inspired chimeric antimicrobials

Nisin (E234), a 32 residue lanthionine-containing peptide, is a potent multifunctional antimicrobial, widely used as a food preservative and commercially developed by Danisco UK. Discovered in 1928, it has gained worldwide acceptance as safe and effective agent in the control of growth and sporulation of food spoilage bacteria and has been in industrial use since the 1950's. Nisin has focused much recent attention as it was shown to act on a highly conserved bacterial pathway by forming membrane binary pores in combination with bacterial cell wall intermediate lipid II (Bonev et al., FASEB J 2004), to inhibit cell wall biosynthesis (Wiedemann et al., JBC 2001; Bonev et al., FASEB J 2004), deregulate bacterial division (Hyde et al., PNAS 2006), peptidoglycan adhesion to membranes and cell wall morhogenesis (Hyde et al., PNAS 2006). In addition to lipid II, undecaprenyl pyrophosphate was identified as another target for nisin and the pyrophosphate group was shown to play crucial role in target recognition (Bonev et al., FASEB J 2004). The N-terminal fragment, nisin1-12, contains the pyrophosphate target recognition motif (Hsu et al., Nat. Struct. Mol. Biol. 2004), while nisin13-34 participates in transmembrane pore-formation. The length of nisin fragment 1-12 is insufficient to allow peptide translocation across the hydrophobic region of bacterial plasma membranes and nisin1-12 cannot form lytic membrane pores alone. At present, we investigate the possibility of replacing the target recognition motif nisin 1-12 with a disulphide-stabilised cystine analogues using molecular dynamics simulations (HPC studentship). Magainin is a 23 residue amphipathic cationic peptide derived from defence secretions of the frog Xenopus laevis (Zasloff, PNAS 1987). It antimicrobial action relies on binding to negatively charged membranes, in which it forms lytic oligomeric pores. The molecule of magainin shows both axial and radial amphipathicity, which allows N-terminus-led membrane insertion followed by formation of non-selective transmembrane pores. In the proposed project we will engineer chimeric molecules carrying N-terminal pyrophosphate-targeting motif, nisin1-12, or a structurally related disulphide analogue, followed by a substitution of the pore-forming section 13-34 for a region structurally derived from magainin. These molecules are intended to offer flexible design and production, broader spectrum of activity, enhanced pore-forming activity and target selectivity. Chimeric molecules based on nisin1-12 and vancomycin have been engineered with activity against VRE (Arnusch et al., Biochemistry 2008). The student will work at Danisco to generate expression systems for the chimerics using the molecular biology base and expertise at the Braband Research Centre and in collaboration with Aalborg University under the supervision of TR, COB and EBH. Structural analysis and target specificity for lipid II and 11PP will be assayed by solid sate NMR with complementary solution NMR in membrane mimics at Nottingham under the supervision of BB and with co-supervision from R. Wimmer at Aalborg for the solution NMR structural studies.