The cytotoxic effect of microcin E492, a low-molecular-mass channel-forming bacteriocin (7,887 Da) produced by a strain of Klebsiella pneumoniae , was characterized in HeLa cells. At low (5 μg/ml) and intermediate (10 μg/ml) concentrations, microcin E492 induced biochemical and morphological changes typical of apoptosis, such as cell shrinkage, DNA fragmentation, extracellular exposure of phosphatidylserine, caspase activation, and loss of mitochondrial membrane potential. Treatment with zVAD-fmk, a general caspase inhibitor, completely blocked the cytotoxic effect of this bacteriocin. At higher microcin concentrations (>20 μg/ml) a necrotic phenotype was observed. Induction of apoptosis by microcin E492 was associated with the release of calcium from intracellular stores, probably after microcin-triggered ion channel formation. Microcin E492 also presented a cytotoxic effect on Jurkat and RJ2.25 cells, but had no effect on KG-1 cells nor on a primary culture of human tonsil endothelial cells, suggesting that there is a specific interaction of the bacteriocin with components of the target cell surface. This report describes a bacteriocin that has the capacity to induce apoptosis in human cell lines.

Microcins are a family of low-molecular weight bacteriocins produced and secreted by Gram-negative bacteria. This review is focused on microcin E492, a pore-forming bacteriocin produced by Klebsiella pneumoniae RYC492 that exerts its antibacterial action on related strains. The steps necessary for the production of active microcin E492 involve post-translational modification with a catechol-type siderophore at the C-terminal and proteolytic processing during export to the extracellular space. This bacteriocin has a modular structure, with a toxic domain at the N-terminal and an uptake domain at the C-terminal of the mature protein. The mechanism by which the C-terminal of microcin E492 is recognized by catecholate siderophore receptors is called the “Trojan horse” strategy, because the C-terminal structure mimics essential bacterial elements, which are recognized by the respective receptors and translocated across the outer membrane to exert antibacterial action. The C-terminal uptake module can be exchanged and used with other toxic domains. Microcin E492 also has a cytotoxic effect on malignant human cell lines. The cytotoxic mechanism is through apoptosis, a desired mechanism for cancer therapy. The ability of microcin E492 to form amyloid-like fibrils constitutes a property that can be exploited in the formulation of this bacteriocin as an antitumoral agent, because these fibrils can behave as stable depots to ensure the sustained release of a biologically active molecule. Alternatively, live bacteria can be used as a continuous source of microcin E492 production in specific tumors.

Microcin E492 is a low‐molecular‐weight, channel‐forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post‐translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.

One of the approaches to address cancer treatment is to develop new drugs not only to obtain compounds with less side effects, but also to have a broader set of alternatives to tackle the resistant forms of this pathology. In this regard, growing evidence supports the use of bacteria-derived peptides such as bacteriocins, which have emerged as promising anti-cancer molecules. In addition to test the activity of these molecules on cancer cells in culture, their in vivo antitumorigenic properties must be validated in animal models. Although the standard approach for such assays employs experiments in nude mice, at the initial stages of testing, the use of high-throughput animal models would permit rapid proof-of-concept experiments, screening a high number of compounds, and thus increasing the possibilities of finding new anti-cancer molecules. A validated and promising alternative animal model are zebrafish larvae harboring xenografts of human cancer cells. Here, we addressed the anti-cancer properties of the antibacterial peptide microcin E492 (MccE492), a bacteriocin produced by Klebsiella pneumoniae, showing that this peptide has a marked cytotoxic effect on human colorectal cancer cells in vitro. Furthermore, we developed a zebrafish xenograft model using these cells to test the antitumor effect of MccE492 in vivo, demonstrating that intratumor injection of this peptide significantly reduced the tumor cell mass. Our results provide, for the first time, evidence of the in vivo antitumoral properties of a bacteriocin tested in an animal model. This evidence strongly supports the potential of this bacteriocin for the development of novel anti-cancer therapies.

Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mytomicin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least 7 protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified a total of 47 asn-tDNA-associated GIs that were classified into 12 groups of homology differing in the encoded functionalities but sharing with GIE492 a conserved recombination module and potentially its mobility features. Most of these GIs encoded factors with proven or potential role in pathogenesis, constituting a major reservoir of virulence factors in this species.

Microcin E492 uptake by FepA, Fiu, and Cir is cooperative, with FepA being the main receptor. No TonB-mediated interaction with the ferric catecholate receptors is needed for microcin to exert action at the cytoplasmic membrane. Microcin E492 uptake by the receptors is inhibited by the dimer and trimer of dihydroxybenzoylserine.