Microcin J25

Multidrug resistant (MDR) bacterial infection has emerged, raising concerns about untreatable infections, and posing the highest health risks. Antimicrobial peptides (AMPs) are thought to be the best remedy for this problem. Here, we showed biosynthetic microcin J25 (MccJ25) exhibited excellent bactericidal activity against standard and clinically relevant veterinary MDR strains with high stability, no cytotoxicity, and no increase in drug resistance. Analysis of antimicrobial mechanism possessed by sensitive enterotoxigenic Escherichia coli (ETEC) based on electron microscopy and Sytox Green methods was carried out. Results showed excellent activity against ETEC was due to permeabilizing bacterial membranes and strong affinity. MccJ25 exhibited high endotoxin-neutralizing activity in both in vivo and in vitro environments, and mice exposed to lipopolysaccharide (LPS) showed decreased plasma LPS levels and improved survival after administration of MccJ25. In an LPS-treated mouse septicemia model, MccJ25 treatment significantly alleviated inflammatory responses by inhibiting proinflammatory factor secretion and expression. In a mouse E. coli infection model, administration of MccJ25 effectively improved host defense against clinically source cocktail of multidrug-resistant E. coli strains induced intestinal inflammation and bacteria dissemination. Results of studies on anti-inflammatory mechanisms showed that MccJ25 downregulated nuclear factor kappa B kinase and mitogen-activated protein kinase, thereby reducing the production of toll-like receptor 4, myeloid differentiation factor 88 and decreasing the key proinflammatory cytokines. These findings clarify MccJ25 may be an ideal antibacterial/antiendotoxic drug candidate that has the potential to further guide the development of anti-inflammatory and/or antimicrobial agents in the war against MDR bacterial infection.

Microcin J25 (MccJ25) is a cyclic antibacterial peptide secreted by a fecal isolate of Escherichia coli. It exerts highly potent activity on Salmonella and Escherichia species. The microcin is recognized at the outer membrane of sensitive strains by the FhuA multifunctional protein, which belongs to the iron/siderophore receptor family, and inhibits bacterial transcription through binding to the RNA-polymerase beta' subunit. The mcjABCD genetic system carried by the wild type 50-kb pTUC100 plasmid contains four genes involved in MccJ25 production and immunity. MccJ25 results from the proteolytic cleavage of a 58-residue precursor at a specific Lys-Gly bond. The resulting mature peptide consists of 21 unmodified amino acids, mostly hydrophobic and includes a single dehydration. The initially described macrocyclic structure of MccJ25, which mostly relied on manual Edman sequencing of the thermolysin-cleaved form (t-MccJ25), involved a head-to-tail cyclisation of the 21-residue precursor. This structure did not prove to be consistent with recent IT-MS CID experiments conducted either on the native microcin or on peptides resulting from acidic or enzymatic cleavages, which are in favour of an 8-residue ring followed by a 13-residue tail. Cyclisation thus occurs between the N-terminus (Gly1) and the Glu8 side chain carboxyl group. The solution three-dimensional structure shows threading of the tail into the ring, thus forming a highly stable lasso type structure. Such a structure was described previously for enzyme inhibitors from Actinobacteria and is consistent with the ability of MccJ25 to inhibit RNA polymerase. The lasso structure is discussed in terms of phylogenetical and biotechnological perspectives.

The increased prevalence of Salmonella spp. resistance in swine spurs the search for alternatives to antibiotics. Microcin J25 (MccJ25), a bacteriocin produced by Escherichia coli, is a potent inhibitor of several pathogenic bacteria including Salmonella enterica. In this study, we aimed to evaluate in vitro the impact of MccJ25 on the composition and the metabolic activity of the swine colonic microbiota. The PolyFermS in vitro continuous fermentation model was used here with modified Macfarlane medium to simulate the porcine proximal colon. During 35 days of fermentation, a first-stage reactor containing immobilized swine fecal microbiota fed two second-stage control and test reactors operated in parallel and used to test the effects of MccJ25 on the composition and the metabolic activity of the microbiota. Reuterin, a broad-spectrum antimicrobial compound produced by Limosilactobacillus reuteri, a lactic acid bacterium naturally present in the gastro-intestinal tract of human and animals, and the antibiotic rifampicin were tested for comparison. Sequencing of 16S rRNA was performed using the Illumina MiSeq technology to evaluate microbial diversity, and liquid chromatography coupled to mass spectrometry (LC-MS) followed by multivariate analysis was used to assess the bacteriocin/antibiotic degradation products and to monitor changes in the swine colonic microbiota metabolome. The results show that MccJ25 or reuterin treatments only induce subtle changes of both the microbial diversity and the metabolome of the swine colon microbiota, while rifampicin induces significant modification in amino acid levels. Although these findings need being validated in vivo, this study affords a first proof of concept for considering MccJ25 as a possible alternative to antibiotics for veterinary and farming applications, taking into account its pathogen-selective and potent inhibitory activity.