Insight into invertebrate defensin mechanism of action: oyster defensins inhibit peptidoglycan biosynthesis by binding to lipid II.
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.
Schmitt
Paulina
P
CNRS, Ifremer, IRD, and Université Montpellier 2, UMR 5119, Laboratoire Ecosystèmes Lagunaires, Place Eugène Bataillon, CC80, 34095 Montpellier, France.
Wilmes
Miriam
M
Pugnière
Martine
M
Aumelas
André
A
Bachère
Evelyne
E
Sahl
Hans-Georg
HG
Schneider
Tanja
T
Destoumieux-Garzón
Delphine
D
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D002855:Chromatography, Thin Layer; D023082:Defensins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D006094:Gram-Positive Bacteria / Q000187:drug effects; D007448:Invertebrates / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D010125:Ostreidae / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D023082:Defensins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol