Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.

Engineering and characterization of human β-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human β-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn’t have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-l-methionyl-l-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

Inhibitory Activity of Natural Synergetic Antimicrobial Consortia Against Salmonella enterica on Broiler Chicken Carcasses

The currently most utilized antimicrobial agent in poultry processing facilities is peracetic acid, a chemical increasingly recognized as hazardous to human health. We evaluated the efficacy of mixtures of natural antimicrobial compounds, namely reuterin, microcin J25, and lactic acid, for reducing the viability of Salmonella enterica and total aerobes on broiler chicken carcasses. The compounds were compared singly and in combination with water and 0.1% peracetic acid. The minimum inhibitory concentrations of reuterin, lactic acid, and microcin J25 against S. enterica serovar Enteritidis were respectively 2 mM, 0.31%, and 0.03 μM. In vitro, the combinations of reuterin + lactic acid and reuterin + microcin J25 were synergic, making these compounds effective at four times lower concentrations than those used alone. Salmonella viable counts fell to zero within 10 min of contact with reuterin + lactic acid at 10 times the concentrations used in combination, compared to 18 h in the case of reuterin + microcin J25. Sprayed onto chilled chicken carcasses, this reuterin + lactic acid mixture reduced Salmonella spp. counts by 2.02 Log CFU/g, whereas reuterin + microcin J25 and peracetic acid reduced them by respectively 0.83 and 1.13 Log CFU/g. The synergy of reuterin with lactic acid or microcin J25 as inhibitors of bacterial growth was significant. Applied as post-chill spray, these mixtures could contribute to food safety by decreasing Salmonella counts on chicken carcasses.

Lasso Peptide Microcin J25 Effectively Enhances Gut Barrier Function and Modulates Inflammatory Response in an Enterotoxigenic Escherichia coli-Challenged Mouse Model

Bacterial resistance leads to severe public health and safety issues worldwide. Alternatives to antibiotics are currently needed. A promising lasso peptide, microcin J25 (MccJ25), is considered to be the best potential substitute for antibiotics to treat pathogen infection, including enterotoxigenic Escherichia coli (ETEC). This study evaluated the efficacy of MccJ25 in the prevention of ETEC infection. Forty-five female BALB/c mice of clean grade (aged seven weeks, approximately 16.15 g) were randomly divided into three experimental groups as follows: (i) control group (uninfected); (ii) ETEC infection group; (iii) MccJ25 + ETEC group. Fifteen mice per group in five cages, three mice/cage. MccJ25 conferred effective protection against ETEC-induced body weight loss, decrease in rectal temperature and increase in diarrhea scores in mice. Moreover, in ETEC-challenged mice model, MccJ25 significantly improved intestinal morphology, decreased intestinal histopathological scores and attenuated intestinal inflammation by decreasing proinflammatory cytokines and intestinal permeability, including reducing serum diamine oxidase and D-lactate levels. MccJ25 enhanced epithelial barrier function by increasing occludin expression in the colon and claudin-1 expression in the jejunum, ultimately improving intestinal health of host. MccJ25 was further found to alleviate gut inflammatory responses by decreasing inflammatory cytokine production and expression via the activation of the mitogen-activated protein kinase and nuclear factor κB signaling pathways. Taken together, the results indicated that MccJ25 protects against ETEC-induced intestinal injury and intestinal inflammatory responses, suggesting the potential application of MccJ25 as an excellent antimicrobial or anti-inflammation agent against pathogen infections.

Effect of Antimicrobial Peptide Microcin J25 on Growth Performance, Immune Regulation, and Intestinal Microbiota in Broiler Chickens Challenged with Escherichia coli and Salmonella

The purpose of this study was to investigate the effects of antimicrobial peptide microcin J25 (MccJ25) on growth performance, immune regulation, and intestinal microbiota in broilers. A total of 3120 one-day-old male Arbor Acres (AA) broilers were randomly allocated to five groups (12 replicates, 52 chickens per replicate). The treatments were control, challenge (0 mg/kg MccJ25), different dosages of antimicrobial peptide (AMP) (0.5 and 1mg/kg MccJ25), and antibiotic groups (20 mg/kg colistin sulfate). The MccJ25 groups increased the body weight gain (starter and overall) that was reduced in the challenge group. The overall (day 1 to day 42) feed-to-gain ratio (G:F) was significantly decreased in AMP groups compared with the challenge group. Birds fed AMP had a decreased population of total anaerobic bacteria (day 21 and day 42) and E. coli (day 21 and day 42) in feces, as well as a lower Salmonella infection rate (day 21 and day 42) compared with birds in the challenge group. The villus height of the duodenum, jejunum, and ileum, as well as the villus height/crypt depth of the duodenum and jejunum were greater in AMP groups than birds in the challenge group. Moreover, MccJ25 linearly improved the villus height of the duodenum and jejunum. The addition of MccJ25 decreased the concentration of TNF-α, IL-1β, and IL-6 compared with challenge group. At d 21, MccJ25 linearly reduced the level of IL-6. In conclusion, dietary supplemented MccJ25 effectively improved performance, systematic inflammation, and improved fecal microbiota composition of the broilers.