Augusto Bellomio

Microcin J25 Has Dual and Independent Mechanisms of Action in Escherichia coli: RNA Polymerase Inhibition and Increased Superoxide Production

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O(2)(*-)) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.

A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria.

Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram‐positive while microcins are active against Gram‐negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries.

Microcin J25 induces the opening of the mitochondrial transition pore and cytochrome c release through superoxide generation

Microcin J25, an antimicrobial lasso‐structure peptide, induces the opening of mitochondrial permeability transition pores and the subsequent loss of cytochrome c. The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction. An increased uptake of calcium is also involved in this process. Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclosporin A and Mn2+, allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c.

Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues.

In this paper we compared the antibacterial activity of native microcin J25, a peptide antibiotic, with the activities of two analogues obtained by chemical modifications. In the first analogue, the negative charge of glutamic carboxyl group was specifically blocked with an L-glycine methyl ester and in the second the histidine imidazole ring was carbethoxylated. Both analogues decreased notably its antibiotic activity against Escherichia coli and Salmonella newport, strains sensible to the native microcin J25. The biological activity of the carbethoxylated analogue was completely recovered after treatment with hydroxylamine. The extreme importance of both polar residues could be interpreted as specific structural features indispensable for the peptide transportation into the cell, extrusion outside the cell or alternatively to inhibit the RNA-polymerase.

Microcin J25 triggers cytochrome c release through irreversible damage of mitochondrial proteins and lipids

We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative-apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.

Tyrosine 9 is the key amino acid in microcin J25 superoxide overproduction.

Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid residues (G(1)-G-A-G-H(5)-V-P-E-Y-F(10)-V-G-I-G-T(15)-P-I-S-F-Y(20)-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr(9) or Tyr(20) with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr(9), and not Tyr(20), is involved in the MccJ25 action on the respiratory chain target.

Inhibitory Effect of the Hybrid Bacteriocin Ent35-MccV on the Growth of Escherichia coli and Listeria monocytogenes in Model and Food Systems

Bacteriocins are being used as new food biopreservative agents. In general, bacteriocins produced by Gram-positive bacteria are active against other Gram-positive. Basically, the same principle applies to those produced by Gram-negative bacteria. They have a restricted spectrum of action against related bacteria to those that produce the bacteriocin. Therefore, other hurdles or chemical preservatives are necessary to apply to broaden the spectrum of action of bacteriocins in foods. This is a further and deeper study of the possible application of the hybrid wide-spectrum bacteriocin named Ent35-MccV in food. Its antimicrobial activity was assayed in skim milk and patties as food models against Listeria monocytogenes and Escherichia coli. The influence of the temperature and digestive proteases on its biological activity and its antimicrobial activity was tested in vitro on a variety of pathogenic and food spoilage bacteria. The results showed that Ent35-MccV could inhibit the growth of both the Gram-positive L. monocytogenes and the Gram-negative E. coli in model food, and its activity was not affected by heating conditions including autoclaving. E. coli strains and Listeria spp. are the most affected bacteria, but Ent35-MccV showed antimicrobial activity against some strain of Salmonella spp., Staphylococcus epidermidis, Enterobacter aerogenes, Morganella morgani, Proteus mirabilis, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes

BACKGROUND Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. METHODS Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. RESULTS The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. CONCLUSIONS These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. GENERAL SIGNIFICANCE Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.

New insights into enterocin CRL35; mechanism of action and immunity revealed by heterologous expression in Escherichia coli

The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin‐producing cells synthesize an immunity protein that forms an inactive bacteriocin–receptor–immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM‐enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM, E. coli cells depolarize and die. Moreover, co‐expression of the immunity protein prevents the deleterious effect of EtpM‐enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM‐enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor.

Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.