Insight into Invertebrate Defensin Mechanism of Action: OYSTER DEFENSINS INHIBIT PEPTIDOGLYCAN BIOSYNTHESIS BY BINDING TO LIPID II

Paulina Schmitt,Miriam Wilmes,Martine Pugnière,André Aumelas,Evelyne Bachère,Hans-Georg Sahl,Tanja Schneider,Delphine Destoumieux-Garzón

doi:10.1074/jbc.m110.143388

Abstract

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.

Highlights

Antimicrobial peptides (AMPs)3 are effector molecules of the innate immune system
We propose that the residues involved in lipid II binding have been conserved through evolution, and we show that residues conferring improved antibacterial activity to oyster defensins by modifying their charge distribution are under diversifying selection
Minimal Inhibitory Concentrations (MICs) values are expressed as the lowest concentration tested that causes 100% of growth inhibition

Summary

Introduction

Antimicrobial peptides (AMPs) are effector molecules of the innate immune system. Present in virtually all living organ-. The AMPs could display a variety of mechanisms of action, killing microbes by membrane disruption (e.g. pore formation) or by altering metabolic processes such as the septum formation, or the cell wall, nucleic acid, and protein syntheses Defensins are 3–5-kDa AMPs that contain three to four disulfide bridges (4) They are broadly distributed in the animal and plant kingdoms (2). Mammalian defensins (␣- and ␤-defensins) adopt a three-stranded antiparallel ␤-sheet structure, and the group of arthropod and plant defensins are composed of an ␣-helix linked to an antiparallel two-stranded ␤-sheet by disulfide bridges, making the so-called cysteine-stabilized ␣-helix/␤-sheet motif (CS␣␤). Antibacterial defensins, which include mammalian, invertebrate (non lepidopteran), and fungal defensins, can be specific inhibitors of a bacterial biosynthesis pathway. The antibacterial activity of two mammalian and one fungal defensin has been recently shown to result from an inhibition of peptidoglycan biosynthesis (20 –22)

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Conclusion