Free Radical Scavenging Activity Of Edf And Its Effects On E.coli

AimEDF(the extracellular death factor) is a novel linear polypeptide secreted by E. coli and discovered in 2007, with the sequence of NNWNN. EDF participates in maz EF-mediated programmed cell death and acts as a quorum-sensing signal molecule. However, it is unclear how EDF delivers the cell-to-cell signaling, and the action mode and targets are also unknown. In this paper, we will study the targets and mechanism of EDF, and its effects on the survival of E. coli. Methods 1) E. coli were treated by Ampicillin(Amp), and the lyzed. Affinity chromatography and SDS-PAGE were performed to get the bands which pulled by EDF. Tandem mass spectra technique and Mascot searching tools were used to identify the proteins binding to EDF. 2) Omp C was purified by SDS methods. His-GS was purified by Ni+-affinity chromatography. Recombinant His-GDH was expressed and purified by Ni+-affinity chromatography. The effects of EDF and its mutants on GS activity were determined. The effects of EDF on GDH activity were also tested. 3) A series of EDF mutants were synthesized by alanine or glycine substitution. The effects of EDF to scavenge DPPH, superoxide radicals, and hydroxyl radicals were tested. 4) The protection effects of EDF on the damage of lipids, protein, and DNA by hydroxyl radicals were investigated. 5) The anti-bacterial effects of Amp combined with EDF was investigated. Results 1) By searching on-line database based on the peptide finger-print mass results, our results indicated that EDF could bind to outer membrane protein Omp C, glutamine synthetase GS, and glutamic acid dehydrogenase GDH.2) EDF and its mutants could inhibit the activity of GS, but not GDH. 3) EDF could not scavenge DPPH radicals, but superoxide radicals and hydroxyl radicals, especially hydroxyl radicals. 4) EDF could protect protein, lipid, and DNA from the damage of hydroxyl radicals. 5) EDF could inhibit the anti-bacterial effects of Amp. Conclusions 1) EDF might enter the cell through Omp C. 2) EDF could inhibit the activity of GS to decrease the consumption of nitrogen nutrients, but not GDH, which lead to the hypothesis that EDF might kill E. coli by inhibit GS. 3) EDF could scavenge superoxide and hydroxyl radicals, and the tryptophan at position 3 might be the key residue for EDF to scavenge hydroxyl radicals. 4) EDF could dose-dependently protect protein, lipid, and DNA from the damage of hydroxyl radicals. 5) EDF could inhibit the anti-bacterial effects of Amp at physiological concentrations. Considering the paper published in Cell on 2007 reported that one common mechanism of ampicillin to kill bacteria is because of hydroxyl radicals release, we think that EDF could not only act as quorum-sensing factor which leads to the death of E. coli, but also increase the survival of E. coli by scavenging hydroxyl radicals. Above all, our results indicated that EDF might have dual effects of “death” and “survival” during the programmed cell death process of E. coli. By this way, the major population of E. coli will die under stress conditions, but the minor population will survive through the protection of EDF. Such dual effects of EDF might be related to the drug resistance of antibiotics.