Efficacy of microcin J25 in biomatrices and in a mouse model of Salmonella infection (original) (raw)
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The Structure and Biological Aspects of Peptide Antibiotic Microcin J25
Current Medicinal Chemistry, 2009
I-G-T15-P-IS -F-Y20-G), excreted to the medium by an Escherichia coli strain. MccJ25 is active on Gram-negative bacteria related to the producer strain, including some pathogenic strains. The four-plasmid genes mcjABCD, are involved in MccJ25 production: mcjA encodes a 58-residue precursor, mcjB and mcjC codify two processing enzymes required for the in vivo synthesis of the mature peptide and mcjD encodes the immunity protein (McjD), a member of the super family of ABC transporters. Immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level. YojI, a chromosomal protein with ATP-binding-cassette-type exporter homology, is also able to export MccJ25. The E. coli outer membrane protein, TolC, is necessary for MccJ25 secretion mediated by either McjD or YojI. The uptake of MccJ25 is dependent on the outer-membrane receptor FhuA and the four inner-membrane proteins TonB, ExbD, ExbB and SbmA. At least two mechanisms described the action of MccJ25 on the target cells: (1) inhibition of the RNA-polymerase (RNAP) activity by obstructing the secondary channel, and consequently, preventing the access of the substrates to its active sites; and (2) operating on the cell membrane, MccJ25 disrupts the electric potential inhibiting the oxygen consumption in Salmonella enterica. MccJ25 also inhibits oxygen consumption and the respiratory chain enzymes in E. coli throughout the increasing of ROS concentration. Nevertheless the exact mechanism of this phenomenon must be elucidated. The MccJ25 exhibits a prolonged antimicrobial activity in a mouse infection model, suggesting a noteworthy potential for therapeutic uses.
Inhibition of Salmonella enterica serovars by microcin J25
FEMS Microbiology Letters, 2004
Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outermembrane 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.
Antibacterial Activity of the Peptide Microcin J25 Produced by Escherichia coli
Medical Laboratory Journal, 2022
Background and objectives: Bacteriocins are generally active antimicrobial peptides effective against bacteria closely related to the producer. Escherichia coli produce two bacteriocins: colicins and microcins. Microcin J25 (Mcc J25) is an antibacterial peptide that inhibits bacterial transcription by disrupting the nucleotide-uptake channel of bacterial RNA polymerase. The objective of this study was to evaluate antimicrobial activity of MccJ25 produced by the bacteriocinogenic E. coli. Methods: In this experimental study, 120 clinical specimens were selected from private diagnostic laboratories in Isfahan (Iran) in 2020. Antagonistic activity of isolates was tested by adopting agar plug method. Total DNA was extracted from clinical specimens and polymerase chain reaction (PCR) was performed using specific primers for amplification of the complete sequence of MccJ25 gene. Accuracy of the PCR products was confirmed by direct sequencing. Homology analysis was performed by using BLAST. Data were analyzed with Chromasv2.1.1 software. Results: Overall, 120 E. coli strains were isolated from the clinical specimens. The antibiotic activity of Mcc J25 was mainly directed at Enterobacteriaceae, including several pathogenic E. coli strains of which 25 had positive well test samples, and about 5 (20%) of the collected clinical samples that were infected with E. coli had the MccJ25 gene. Conclusions: Based on the results, Mcc J25 has favorable antibacterial potential, which can be further exploited as an alternative to chemical antibiotics.
Research Square (Research Square), 2024
Introduction: Microcins are Antimicrobial peptides (AMPs) with low molecular weight, which are produced by Enterobacterales and have broad-spectrum antibacterial activity. They can selectively replace common cancer treatments in cancer cells with less side effects and higher effectiveness. Given the aforementioned context, the present study endeavors to examine the antitumor activity of microcins isolated from of the Enterobacterales. Material and Methods In total, 120 Enterobacterales isolates were examined after identi cation. Subsequently, the bacteria were subjected to an agar diffusion test to assess their antibacterial e cacy. Positive isolates were further examined for the presence of Mccj25 using PCR. The cytotoxic effects of isolates harboring the microcin gene were explored using quantitative real-time PCR (RT-qPCR) and the MTT test on breast cancer cells. Additionally, the expression levels of BCL2 and STAT3 genes were evaluated, and apoptosis was quanti ed using ow cytometry. The repair rate of normal cells was determined using a scratch assay. Results The ndings obtained from the phenotypic and biochemical assays have duly veri ed and established the categorization of the Enterobacterales. After conducting the agar diffusion test, a total of 25 isolates of Escherichia coli and Klebsiella pneumoniae displaying inhibition zones were chosen as suitable specimens possessing AMPs. Urinary E. coli was identi ed as isolate 83. The analysis conducted on the expression of the Mccj25 gene within the aforementioned isolates indicated that isolate 83 exhibited signi cant expression of the Mccj25 gene. Conclusion The extract obtained from this isolate on the breast cancer cell line exhibited the most signi cant degree of toxicity after precisely 48 h. Furthermore, the treatment of breast cancer cells with isolate 83 showed that the rate of apoptosis was about 86%, and the expression of BCL2 and STAT3 genes decreased. Moreover, it potentiated the reparative ability of normal broblast cells. They resulted in growth suppression of breast cancer cells and elicited an escalated rate of cellular demise via the apoptosis pathway. the use of medicinal drugs with fewer side effects has received considerable attention in recent years. Antimicrobial peptides (AMPs) are a class of anti-cancer peptides. AMPs have a wide range of antibacterial, antiviral, antifungal, and anticancer properties, and are part of the intrinsic resistance to microorganisms [3]. AMPs, members of the class of anticancer peptides, are among these substances. AMPs have a wide range of antibacterial, antiviral, antifungal, and anticancer properties and are part of the intrinsic resistance reaction to microorganisms [3]. Numerous studies have reported the use of AMPs against cancer cells, including breast, lung, lymphoma, leukemia, and myeloma cells [4].. E. coli bacteriocins are divided into colicins (25-80 kDa) and microcins (1-10 kDa) based on their molecular weight [5, 6]. Microcins possess an advantage over colicins as they do not pose lethality to the strains that produce them. [7]. There are two types of microcins. Class I microcins have a molecular mass of less than 5 kDa and are highly post-translationally modi ed, which include microcin B17 (MccB17), MccJ25, MccC7/C51, and MccD93. In contrast, class II peptide microcins are larger (5-10 kDa) and are further divided into two subclasses, IIa and IIb. Accordingly, subclass IIa does not require post-translational modi cations, but can contain disul de bonds, including the plasmid-mediated microcins MccL, MccV, and MccS, and siderophore bacteriocins, known as class IIb microcins, including MccE492, MccM, MccI47, and MccH47 [8]. The signi cance of utilizing microcins as possible alternatives to antibiotics is attributable to the absence of bacterial resistance to these AMPs [9]. In addition to their antimicrobial properties, microcins have antitumor functions and can act through various mechanisms such as apoptosis, necrosis, and pore creation in the target cell membrane, thereby inhibiting angiogenesis and activating the immune system against cancer cells [10]. Recently, researchers have exhibited signi cant interest in the potential use of microcins for cancer treatment because of their selective nature, which results in higher e cacy and minimal adverse effects. [9, 10]. MccJ25 is a 21-amino acid peptide produced by some Enterobacterales, especially E. coli, and has a stable structure to avoid degradation by proteases in the digestive tract [8]. MccJ25 has antimicrobial activity and as a membrane-active peptide, it causes the death of eukaryotic cells by destroying cytochrome c [11, 12]. Against this background, in the current study, we examined how breast cancer cells respond to isolated MccJ25 from E. coli. 2. Materials and Methods 2.1. isolate collection, culture, and identi cation E. coli were isolated and con rmed from urine samples obtained from private laboratories. In brief, 120 Enterobacterales isolates were cultured on blood agar and eosin methylene blue at 35°C for 18-24 h. Next, E. coli isolates were identi ed and isolated using different phenotypic characteristics and biochemical tests, including Voges Proskauer (VP)
Mechanism of Bactericidal Activity of Microcin L in Escherichia coli and Salmonella enterica
Antimicrobial Agents and Chemotherapy, 2011
For the first time, the mechanism of action of microcin L (MccL) was investigated in live bacteria. MccL is a gene-encoded peptide produced by Escherichia coli LR05 that exhibits a strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. We first subcloned the MccL genetic system to remove the sequences not involved in MccL production. We then optimized the MccL purification procedure to obtain large amounts of purified microcin to investigate its antimicrobial and membrane properties. We showed that MccL did not induce outer membrane permeabilization, which indicated that MccL did not use this way to kill the sensitive cell or to enter into it. Using a set of E. coli and Salmonella enterica mutants lacking iron-siderophore receptors, we demonstrated that the MccL uptake required the outer membrane receptor Cir. Moreover, the MccL bactericidal activity was shown to depend on the TonB protein that transduces the proton-motive force of the cytoplasmic membrane to transport iron-siderophore complexes across the outer membrane. Using carbonyl cyanide 3-chlorophenylhydrazone, which is known to fully dissipate the proton-motive force, we proved that the proton-motive force was required for the bactericidal activity of MccL on E. coli. In addition, we showed that a primary target of MccL could be the cytoplasmic membrane: a high level of MccL disrupted the inner membrane potential of E. coli cells. However, no permeabilization of the membrane was detected.
Journal of Bacteriology, 2007
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.
Food and Bioprocess Technology, 2015
Bacteriocins are being used as new food biopreservative agents. In general, bacteriocins produced by Gram-positive bacteria are active against other Grampositive. 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 Grampositive 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.
Sensitization of Microcin J25-Resistant Strains by a Membrane-Permeabilizing Peptide
Applied and Environmental Microbiology, 2010
Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli. MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake into E. colisensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on some E. coli, Salmonella, and Shigella species strains, while other Gram-negative bacteria, such as clinical isolates of Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Moraxella catarrhalis, and Salmonella enterica serovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membranepermeabilizing peptide (KFF) 3 K made some resistant strains sensitive to MccJ25, among them S. Typhimurium, where the antibiotic inhibits in vitro cell growth and bacterial replication within macrophages. The results demonstrate that the membrane permeabilization induced by (KFF) 3 K allows MccJ25 penetration in an FhuA and SbmA-independent manner and suggest that the combination of both peptides could be considered as a therapeutic agent against pathogenic Salmonella strains.