Résumé
Microcins are gene-encoded antibacterial peptides secreted by enterobacteria. Active against phylogenetically related microorganisms, they are potent effectors of the microbial competitions within the intestinal tract. Microcins display a broad diversity of structures and mechanisms of action. Microcin E492 (MccE492) is naturally produced by Klebsiella pneumoniae. Though first isolated as an 7886 Da-unmodified peptide, we have recently shown that it also exists in a posttranslationally modified form (MccE492m, 8717 Da) more potent than MccE492 with MICs in the nanomolar range. MccE492m posttranslational modification was characterized by a series of biochemical and spectroscopic methods. A 1772 Da-fragment purified from MccE492m chymotrypsin digest but absent from MccE492 hydrolysate was shown to contain the entire 831 Da-modification. The complete structure of the modification was identified by subjecting this 1772 Da-peptide to MS fragmentation and 800 MHz NMR analyses. Interestingly, the modification contains a trimer of N-(2,3-dihydroxybenzoyl)-L-serine (DHBS), which belongs to a group of catechol-type siderophores involved in iron uptake by enterobacteria. Moreover, MSn experiments in presence of FeCl3 showed that the modification was able to capture Fe3+. MccE492m is the first example of a novel type of antibacterial peptides that we called siderophore-peptides. The activity of MccE492 and MccE492m was compared. In vitro, both peptides formed ion-channels in planar lipid bilayers. In vivo, they depolarized and permeabilized the cytoplasmic membrane of E. coli. Both the antibacterial and membrane activities of the peptides were TonB- and energy-dependent. The receptors for MccE492 and MccE492m were identified by assaying their antibacterial activity against isogenic bacteria carrying mutations in outer membrane proteins known as iron/siderophore receptors. The three DHBS-receptors FepA, Fiu, and Cir, which are known as TonB-dependent, were shown to be involved in both MccE492 and MccE492m recognition and antimicrobial activity. Since recognition of both MccE492 and MccE492m is mediated by iron/siderophore receptors at the surface of sensitive bacteria, we propose that improvement of MccE492 antimicrobial activity upon modification results from an increase of the microcin/receptor affinity.