oyster defensin variants (membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-pore formation) or by altering metabolic processes such as the septum formation or the cell wall nucleic acid and protein syntheses (for review see Refs. diverse spectra of activity of AMPs are believed to be indicative of different SNT-207707 modes of action (5). However the mechanisms of how defensins kill microorganisms are still incompletely comprehended. It is well established that this amphiphilic structure they adopt is crucial for the first interaction with the microbial surface (11). In addition several defensins have been reported to damage bacterial and artificial membranes including mammalian α- and β-defensins (12 13 as well as arthropod defensins (14 15 However nonmembrane-disruptive mechanisms of action have also been proposed as for the α-defensin HNP-1 which appears to transit across the cytoplasmic membrane with minimal disruption (13). Thus over the past years the debate has increased on how far membrane disruption accounts for the antimicrobial activity of defensins and other AMPs (16 -18). Strictly antifungal defensins which include defensins from plants and from lepidopteran insects are not only membrane-disrupting agents but also interact with fungal glucosylceramides (19). Similarly antibacterial defensins which include mammalian invertebrate (non lepidopteran) and fungal defensins can be specific inhibitors of a bacterial biosynthesis pathway. For instance the antibacterial activity of two mammalian and one fungal defensin has been recently shown to result from an inhibition of peptidoglycan biosynthesis (20 -22). We have performed here a comparative study of the mechanism of action of antibacterial invertebrate defensins the cellular targets of which are still unknown. For that we used as a model three defensin variants characterized in the oyster One was identified from the oyster mantle (and assays including UDP-MurNAc-pp accumulation assays thin layer chromatography surface plasmon resonance and NMR we showed that all oyster defensins inhibit peptidoglycan biosynthesis by Rabbit polyclonal to VAV1.The protein encoded by this proto-oncogene is a member of the Dbl family of guanine nucleotide exchange factors (GEF) for the Rho family of GTP binding proteins.The protein is important in hematopoiesis, playing a role in T-cell and B-cell development and activation.This particular GEF has been identified as the specific binding partner of Nef proteins from HIV-1.Coexpression and binding of these partners initiates profound morphological changes, cytoskeletal rearrangements and the JNK/SAPK signaling cascade, leading to increased levels of viral transcription and replication.. SNT-207707 binding to lipid II. 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. MATERIALS AND METHODS Recombinant Expression of Cg-Defs Recombinant Rosetta (DE3) as an N-terminal His6-tagged fusion protein using the pET-28a system (Novagen). By PCR amplification using the forward SNT-207707 primer 5′-GCGCGAATTCATGGGATTTGGGTGTCCG-3′ paired with reverse primer 5′-ATATATGTCGACCTTGAAAGATCTTTACTTC-3′ a Met-coding trideoxynucleotide was incorporated 5′ of each cDNA of CIP 5345CIP 6620 CIP 103428 SG511 22 (nice gift from P. Bulet) and SBS363. Marine strains were CIP 104228 CIP 105733 ATCC 19264 CIP 103195 and the oyster pathogens CIP 107715 (also known as LGP32) and CIP 102971 (also known as LPi 02/41). MICs were decided in duplicate by the liquid growth inhibition assay based on the procedure described by Hétru and Bulet (25). MIC values are expressed as the lowest concentration tested that causes 100% SNT-207707 of growth inhibition (micromolar). Poor broth (PB: 1% bactotryptone 0.5% NaCl w/v pH 7.5) nutrient medium was used for standard bacteria and artificial sea water (26) supplemented with 4 g/liter bactopeptone and 1 g/liter yeast extract (referred to as Zobell medium) at a third strength was used for marine bacteria. Growth was monitored spectrophotometrically at 620 nm on a Multiscan microplate reader (Labsystems). Antagonization Assays Different peptidoglycan precursors namely undecaprenyl phosphate (C55P) UDP-MurNAc-pp lipid II or UDP-GlcNAc were tested for antagonization of the oyster defensin antimicrobial activity. Basically serial dilutions of defensins were performed from SNT-207707 0.25 to 8× MIC each dilution being SNT-207707 incubated in a microtiter plate with the peptidoglycan precursors in a 1:1 1 or 1:5 molar ratio. SG511 was then added to the microtiter plate as for a conventional MIC determination. Culture medium was half-concentrated Mueller-Hinton broth (Oxoid). After an 18-h incubation at 37 °C the lowest peptide/peptidoglycan precursor molar ratio that antagonized the antimicrobial..