GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Engineering and Purification of Microcin C7 Variants Resistant to Trypsin and Analysis of Their Biological Activity.

Microcin C7 (McC) as a viable form of antimicrobial has gained substantial attention due to its distinctive antimicrobial activity, by targeting aspartyl tRNA synthetase. McC can be a potential solution against pathogenic microbial infections in the postantibiotic era. However, considering that degradation by digestive enzymes can disrupt the function of this peptide in the gastrointestinal tract, in this study, we attempt to design McC variants to overcome several barriers that may affect its stability and biological activity. The mccA gene encoding the McC peptide precursor was mutated and 12 new McC variants with trypsin resistance were found. The Yej+rimL- strain was used as an indicator to determine the minimum inhibitory concentrations (MICs). The results showed that three variants, including R2A, R2T and R2Q, among 12 variants formed by the replacement of the second arginine of the McC peptide with different amino acids, were resistant to trypsin and had an outstanding antimicrobial ability, with MIC values of 12.5, 25, and 25 µg/mL, respectively. Taken together, our findings show that the engineering of the site-directed mutagenesis of McC significantly enhances McC trypsin resistance and maintains a great antimicrobial activity.

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-1ß, 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.

Therapeutic administration of the recombinant antimicrobial peptide microcin J25 effectively enhances host defenses against gut inflammation and epithelial barrier injury induced by enterotoxigenic Escherichia coli infection.

Recombinant antimicrobial peptide microcin J25 (MccJ25) causes potent antimicrobial activity against enterotoxigenic Escherichia coli (ETEC) in vitro; however, independently of this activity, its role in suppressing intestinal inflammation and epithelial barrier injury in vivo remains unclear. We investigated the therapeutic effects of MccJ25 on intestinal inflammation and epithelial barrier dysfunction and the underlying mechanism, using gentamicin for comparison. In a mouse model of intestinal inflammation, therapeutic administration of either MccJ25 or gentamicin after ETEC K88 infection attenuated clinical symptoms, reduced intestinal pathogen colonization, improved intestinal morphology, and decreased inflammatory pathologies and intestinal permeability, ultimately improving the hosts' health. MccJ25 also attenuated ETEC-induced mouse intestinal barrier dysfunction by enhancing tight junction proteins (TJPs). Using the human epithelial cell line Caco-2, we verified the epithelial barrier-strengthening and mucosal injury-alleviating effects of MccJ25 on ETEC infection: increased expression of TJPs by activating the p38/MAPK pathway, balancing the microbiota, and improving short-chain fatty acid concentrations in the cecum of ETEC-infected mice. Although gentamicin and MccJ25 had similar effects in the inflamed gut, MccJ25 was superior to gentamicin with regard to defending the host from ETEC infection. Overall, MccJ25 may be a promising therapeutic drug for treating enteric pathogen-induced intestinal inflammation diseases.

A Comprehensive Antimicrobial Activity Evaluation of the Recombinant Microcin J25 Against the Foodborne Pathogens Salmonella and E. coli O157:H7 by Using a Matrix of Conditions.

Natural microcin J25 (MccJ25) represent promising alternatives to traditional antibiotics for the treatment of drug-resistant infections. However, little is known about the antibacterial activity of recombinant MccJ25 against foodborne pathogens. Here, the activity of recombinant MccJ25 was examined using a matrix of conditions in order to assess the efficacy of recombinant MccJ25 as a mitigation against foodborne pathogens, such as Salmonella species and Escherichia coli (E. coli) O157:H7. Results showed that recombinant MccJ25 displayed excellent antimicrobial activity against these foodborne pathogens, including clinical isolates of Salmonella and E. coli, as well as clinical antibiotic-resistant Salmonella and E. coli isolates with different minimal inhibitory concentrations. In addition, antimicrobial activity curves and Live/Dead assay evidenced that recombinant MccJ25 harbors strong bactericidal activity against Salmonella and E. coli O157:H7. Notably, recombinant MccJ25 also had great potency and induced fast mortality against different growth phase of Salmonella and E. coli. The stability analysis results showed that the activity of recombinant MccJ25 was not influenced by temperatures as high as 121°C. Varying the pH from 2.0 to 9.0 did not appear to affect the activity of recombinant MccJ25. Under the challenge of several proteases, simulated gastrointestinal fluids and serum, recombinant MccJ25 still maintained exceptionally strong antimicrobial activity. Significant reductions in Salmonella Pullorum levels were also achieved in food biological environments, such as milk, egg and meat. Moreover, we demonstrated that recombinant MccJ25 appeared to act by inducing membrane breaks, thinning, and disintegration in the Salmonella Pullorum cytoplasmic membrane. Taken together, these results indicated that recombinant MccJ25 could be an effective alternative for mitigating and prevention of Salmonella and E. coli infection in food, animal and agriculture applications.

Protective Ability of Biogenic Antimicrobial Peptide Microcin J25 Against Enterotoxigenic Escherichia Coli-Induced Intestinal Epithelial Dysfunction and Inflammatory Responses IPEC-J2 Cells.

Poison of intestinal induce severe health problems in human infants and young animals due to contaminating foods and feedstuffs. With the emergence of public health concerns and high-speed diffuse of drug-opposition of bacteria, the adoption of antimicrobial peptides as potential candidates in treating pathogen infections raised up. Nature Microcin J25 (MccJ25), a class of lasso peptides separated from a fecal strain of E. coli, has been replied to display powerful antimicrobial behavior. Herein, the study was to assess the usefulness of biogenic MccJ25 in the prophylaxis of ETEC K88 infection in IPEC-J2 cells. In vitro antimicrobial activity against ETEC K88 and cytotoxicity of biogenic MccJ25 were determined first. To further understand how biogenic MccJ25 mediates its impact, ETEC K88 adhesion in cells, membrane permeability [as indicated by reduced release of lactate dehydrogenase (LDH)], transepithelial electrical resistance (TEER), barrier function, and proinflammatory cytokines levels were determined in IPEC-J2 cells after treatment with biogenic MccJ25 and challenge with ETEC K88. Biogenic MccJ25 had a minimum inhibitory concentration of 0.25 µg/mL against ETEC K88, decreased ETEC K88 adhesion in cells and did not cause cytotoxicity toward cells. Furthermore, biogenic MccJ25 protects against ETEC-induced barrier dysfunction by increasing the TEER, decreasing the LDH and promoting tight junction proteins (TJPs) by promoting the assembly of occludin and claudin-1 in the tight junction complex. Biogenic MccJ25 was further found to relieve inflammation responses through modulation of interleukine-6, IL-8 and tumor necrosis factor-α levels via inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor κB activation. In summary, biogenic MccJ25 can protects against ETEC K88-induced intestinal damage and inflammatory response, recommend the hidden adoption of biogenic MccJ25 as a novel prophylactic agent to reduce pathogen infection in animals, food or humans.