Synthetic peptides derived from the sequence of a lasso peptide microcin J25 show antibacterial activity

Microcin J25 (MccJ25) is a plasmid-encoded, ribosomally synthesized antibacterial peptide with a unique lasso structure. The lasso structure, produced with the aid of two processing enzymes, provides exceptional stability to MccJ25. We report the synthesis of six peptides (1-6), derived from the MccJ25 sequence, that are designed to form folded conformation by disulfide bond formation and electrostatic or hydrophobic interactions. Two peptides (1 and 6) display good activity against Salmonella newport, and are the first synthetic derivatives of MccJ25 that are bactericidal. Peptide 1 displays potent activity against several Salmonella strains including two MccJ25 resistant strains. The solution conformation and the stability studies of the active peptides suggest that they do not fold into a lasso conformation and peptide 1 displays antimicrobial activity by inhibition of target cell respiration. Like MccJ25, the synthetic MccJ25 derivatives display minimal toxicity to mammalian cells suggesting that these peptides act specifically on bacterial cells.     

Redox-active tyrosine residue in the microcin J25 molecule

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal ‘tail’ passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H₂O₂ was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.     

Protective action of ppGpp in microcin J25-sensitive strains

As Escherichia coli strains enter the stationary phase of growth they become more resistant to the peptide antibiotic microcin J25. It is known that starvation for nutrients such as amino acids or glucose leads to increases in guanosine 3',5'-bispyrophosphate (ppGpp) levels and that the intracellular concentration of this nucleotide increases as cells enter the stationary phase of growth. Therefore, we examined the effects of artificially manipulating the ppGpp levels on sensitivity to microcin J25. A direct correlation was found between ppGpp accumulation and microcin resistance. Our results indicate that the nucleotide is required to induce production of YojI, a chromosomally encoded efflux pump which, in turn, expels microcin from cells. This would maintain the intracellular level of the antibiotic below a toxic level.     

Inhibition of Salmonella enterica serovars by microcin J25

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outer-membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies.     

Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25

Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.     

Antimitochondrial activity displayed by the antimicrobial peptide microcin J25

In this report we studied the effect of the antimicrobial peptide, microcin J25, on the rat heart mitochondria. This peptide induced an important inhibition of the ATP synthesis with the concomitant enhancement of the ATP degradation. These effects were the result of two processes: on one hand, microcin J25 was able to insert into the membrane and hence alter its permeability with the consequent dissipation of the proton motive force. On the other, microcin J25 inhibited the enzymatic activity of the cytochrome c reductase (complex III) of the respiratory chain. The relevance of this study to the potential use of microcin J25 as an anti-tumoral agent is discussed.     

Antibacterial activity evaluation of microcin J25 against diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 microg ml(-1), respectively. A 10(4)-CFU ml(-1) contamination by this strain was destroyed in milk and meat extract by 6.25 microg of J25 ml(-1) and in half-diluted egg yolk by 50 microg of J25 ml(-1).     

Genetic analysis of plasmid determinants for microcin J25 production and immunity.

Microcin J25 (MccJ25) is a small peptide antibiotic produced by an Escherichia coli strain isolated from human feces. The genetic determinants for MccJ25 synthesis and immunity have been cloned from the low-copy-number wild-type plasmid pTUC1OO into the compatible vectors pBR322 and pACYC184. Physical and phenotypical analysis of insertion mutations and complementation tests defined three contiguous genes involved in MccJ25 production which span a region of about 2.2 kb. Immunity to the antibiotic is provided by an additional gene adjacent to the production region.     

YojI of Escherichia coli functions as a microcin J25 efflux pump

In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.     

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 (G¹-G-A-G-H⁵-V-P-E-Y-F¹⁰-V-G-I-G-T¹⁵-P-I-S-F-Y²⁰-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 Tyr⁹ or Tyr²⁰ 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 Tyr⁹, and not Tyr²⁰, is involved in the MccJ25 action on the respiratory chain target.     

Phenomic and genomic approaches to studying the inhibition of multiresistant Salmonella enterica by microcin J25

In livestock production, antibiotics are used to promote animal growth, control infections and thereby increase profitability. This practice has led to the emergence of multiresistant bacteria such as Salmonella, of which some serovars are disseminated in the environment. The objective of this study is to evaluate microcin J25 as an inhibitor of Salmonella enterica serovars of various origins including human, livestock and food. Among the 116 isolates tested, 37 (31.8%) were found resistant to at least one antibiotic, and 28 were multiresistant with 19 expressing the penta-resistant phenotype ACSSuT. Microcin J25 inhibited all isolates, with minimal inhibitory concentration values ranging from 0.06 μg/ml (28.4 nM) to 400 μg/ml (189 μM). Interestingly, no cross-resistance was found between microcin J25 and antibiotics. Multiple sequence alignments of genes encoding for the different proteins involved in the recognition and transport of microcin J25 showed that only ferric-hydroxamate uptake is an essential determinant for susceptibility of S. enterica to microcin J25. Examination of Salmonella strains exposed to microcin J25 by transmission electronic microscopy showed for the first-time involvement of a pore formation mechanism. Microcin J25 was a strong inhibitor of several multiresistant isolates of Salmonella and may have a great potential as an alternative to antibiotics.     

Inhibition of pathogenic Salmonella enteritidis growth mediated by Escherichia coli microcin J25 producing strains

For the first time, microcin-producing strains showing inhibitory activities against enteropathogen Salmonella enteritidis were isolated from poultry intestinal contents. Among the numerous strains isolated, two strains of Escherichia coli, named J02 and J03, showing the greatest activities against S. enteritidis, were studied. Biochemical tests and purification identified the main antagonist compound produced as microcin J25. In order to evaluate the protective potential of E. coli J02 and J03 against S. enteritidis infection, the ability of these strains to inhibit growth of S. enteritidis was investigated in mixed culture. A strong antagonist activity was obtained with a preculture phase of the active strain in minimal medium before incubation with S. enteritidis. In a bioreactor experiment simulating the chicken gastric and intestinal tract environment, a mixture of the two strains E. coli J02 and J03, provided an enhanced inhibitory effect. Microcinogenic strain activities were not affected by bile, pancreatic enzymes addition, or acidic conditions. These results suggest the relevant role of microcin-producing microorganisms in microbial intestinal ecology. To conclude, this study shows that microcin J25 strains could exert a beneficial protective effect against S. enteritidis growth in situ.     

Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25

A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.     

Solution structure of microcin J25, the single macrocyclic antimicrobial peptide from Escherichia coli.

The three-dimensional solution structure of microcin J25, the single cyclic representative of the microcin antimicrobial peptide class produced by enteric bacteria, was determined using two-dimensional 1H NMR spectroscopy and molecular modeling. This hydrophobic 21-residue peptide exhibits potent activity directed to Gram-negative bacteria. Its primary structure, cyclo(-V1GIGTPISFY10GGGAGHVPEY20F-), has been determined previously [Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R.A., Farías, R.N., Moreno, F. & Rebuffat, S. (1999) Eur. J. Biochem., 259, 747-755]. Conformational parameters (3JNHCalphaH coupling constants, quantitative nuclear Overhauser enhancement data, chemical shift deviations, temperature coefficients of amide protons, NH-ND exchange rates) were obtained in methanol solution. Structural restraints consisting of 190 interproton distances inferred from NOE data, 11 phi backbone dihedral angle and 9 chi1 angle restraints derived from the coupling constants and three hydrogen bonds in agreement with the amide exchange rates were used as input for simulated annealing calculations and energy minimization in the program XPLOR. Microcin J25 adopts a well-defined compact structure consisting of a distorted antiparallel beta sheet, which is twisted and folded back on itself, thus resulting in three loops. Residues 7-10 and 17-20 form the more regular part of the beta sheet. The region encompassing residues Gly11-His16 consists of a distorted beta hairpin, which divides into two small loops and is stabilized by an inverse gamma turn and a type I' beta turn. The reversal of the chain leading to the Phe21-Pro6 loop results from a mixed beta/gamma turn. A cavity, in which the hydrophilic Ser8 side-chain is confined, is delimited by two crab pincer-like regions that comprise residues 6-8 and 18-1.     

Thermolysin-linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity.

Microcin J25 (MccJ25) is the single macrocyclic antimicrobial peptide belonging to the ribosomally synthesized class of microcins that are secreted by Enterobacteriaceae. It showed potent antibacterial activity against several Salmonella and Escherichia strains and exhibited a compact three-dimensional structure [Blond et al. (2001) Eur. J. Biochem., 268, 2124-2133]. The molecular mechanisms involved in the biosynthesis, folding and mode of action of MccJ25 are still unknown. We have investigated the structure and the antimicrobial activity of thermolysin-linearized MccJ25 (MccJ25-L1-21: VGIGTPISFY10GGGAGHVPEY20F), as well as two synthetic analogs, sMccJ25-L1-21 (sequence of the thermolysin-cleaved MccJ25) and sMccJ25-L12-11 (C-terminal sequence of the MccJ25 precursor: G12GAGHVPEYF21V1GIGTPISFYG11). The three-dimensional solution structure of MccJ25-L1-21, determined by two-dimensional NMR, consists of a boot-shaped hairpin-like well-defined 8-19 region flanked by disordered N and C termini. This structure is remarkably similar to that of cyclic MccJ25, and includes a short double-stranded antiparallel beta-sheet (8-10/17-19) perpendicular to a loop (Gly11-His16). The thermolysin-linearized MccJ25-L1-21 had antibacterial activity against E. coli and S. enteritidis strains, while both synthetic analogues lacked activity and organized structure. We show that the 8-10/17-19 beta-sheet, as well as the Gly11-His16 loop are required for moderate antibacterial activity and that the Phe21-Pro6 loop and the MccJ25 macrocyclic backbone are necessary for complete antibacterial activity. We also reveal a highly stable 8-19 structured core present in both the native MccJ25 and the thermolysin-linearized peptide, which is maintained under thermolysin treatment and resists highly denaturing conditions.     

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.     

An experimental and computational investigation of the post-yield behaviour of trabecular bone during vertebral device subsidence

Interbody fusion device subsidence has been reported clinically. An enhanced understanding of the mechanical behaviour of the surrounding bone would allow for accurate predictions of vertebral subsidence. The multiaxial inelastic behaviour of trabecular bone is investigated at a microscale and macroscale level. The post-yield behaviour of trabecular bone under hydrostatic and confined compression is investigated using microcomputed tomography-derived microstructural models, elucidating a mechanism of pressure-dependent yielding at the macroscopic level. Specifically, microstructural trabecular simulations predict a distinctive yield point in the apparent stress–strain curve under uniaxial, confined and hydrostatic compression. Such distinctive apparent stress–strain behaviour results from localised stress concentrations and material yielding in the trabecular microstructure. This phenomenon is shown to be independent of the plasticity formulation employed at a trabecular level. The distinctive response can be accurately captured by a continuum model using a crushable foam plasticity formulation in which pressure-dependent yielding occurs. Vertebral device subsidence experiments are also performed, providing measurements of the trabecular plastic zone. It is demonstrated that a pressure-dependent plasticity formulation must be used for continuum level macroscale models of trabecular bone in order to replicate the experimental observations, further supporting the microscale investigations. Using a crushable foam plasticity formulation in the simulation of vertebral subsidence, it is shown that the predicted subsidence force and plastic zone size correspond closely with the experimental measurements. In contrast, the use of von Mises, Drucker–Prager and Hill plasticity formulations for continuum trabecular bone models lead to over prediction of the subsidence force and plastic zone.     

The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.