Fluorescence Microscopy Monitoring of Nisin Binding to Bactoprenol Bound Cell Wall Precursors

Abstract

The antimicrobial peptide nisin displays its activity by a unique dual mechanism. It forms pores within cell membranes of Gram-positive bacteria by binding to the cell wall precursor lipid II and inhibits the cell wall synthesis [1]. We examined membrane-nisin interactions by confocal microscopy of giant unilamellar vesicles (GUVs) similarly as recently for TAT peptides [2].Interaction of nisin with pure phospholipid GUVs was controlled by electrostatic attraction. Presence of lipid II in the GUV membrane elicited a quite different response. When 0.2 mol% lipid II were integrated in phosphatidylcholine membranes, specific interactions with lipid II led to immediate binding of nisin and formation of large lipid II:nisin aggregates. Simultaneously, pore formation occurred. The same effects of specific binding were observed with lipid I, the precursor of lipid II in the cell wall biosynthesis cycle.In contrast, none of these effects was observed for GUV membranes containing undecaprenyl-phosphate (C55-P) and undecaprenyl-diphosphate (C55-PP), the carrier lipids for the cell wall building block. This finding was according to expectation for C55-P, but unexpected for C55-PP since its pyrophosphate unit was previously described as nisin binding site [3]. Our results show that the interaction of nisin with lipid II resulting in aggregation and pore formation requires further structural elements in addition to the bactoprenoldiphosphate unit.[1] H. E. Hasper, N. E. Kramer, J. L. Smith, J. D. Hillman, C. Zachariah, O. P. Kuipers, B. de Kruijff, E. Breukink, Science 2006, 313, 1636.[2] C. Ciobanasu, J.-P. Siebrasse, U. Kubitscheck, Biophys. J. 2010, 99, 153.[3] S.-T. D. Hsu, E. Breukink, E. Tischenko, M. A. G. Lutters, B. de Kruijff, R. Kaptein, A. M. J. J. Bovin, N. A. J. van Nuland, Nat. Struct. Mol. Biol., 2004, 11, 963.