A novel glycine-rich peptide from Zophobas atratus, coleoptericin B, targets bacterial membrane and protects against Klebsiella pneumoniae-induced mastitis in mice.

OBJECTIVES: The growing occurrence of bacterial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides, a class of small molecules with antimicrobial activity, have been regarded as the ideal alternatives to antibiotics. METHODS: In this study, we amplified a new type of Zophobas atratus coleoptericin (denoted coleoptericin B) through rapid amplification of cDNA ends (RACE) PCR and expressed recombinant Z. atratus coleoptericin B (rZA-col B) by prokaryotic expression. Subsequently, we evaluated the antimicrobial effect and biocompatibility of rZA-col B in vivo, investigated its antimicrobial mechanism, and assessed its therapeutic effect in a murine model of mastitis caused by MDR Klebsiella pneumoniae. RESULTS: The in vivo studies demonstrated that rZA-col B possesses broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. It exhibited less than 1.5% haemolysis and 10% cytotoxicity, even at a concentration of 128 µM. Additionally, rZA-col B had a minimal risk of inducing drug resistance. Furthermore, rZA-col B could disrupt the integrity of bacterial membranes, induce membrane permeabilization and ultimately lead to bacterial death. Importantly, rZA-col B also alleviated mastitis caused by MDR K. pneumoniae in a murine model by enhancing bacterial clearance, reducing neutrophil infiltration, decreasing TNF-α and IL-1ß expression, and protecting the mammary barrier. CONCLUSIONS: rZA-col B may be a promising antibacterial agent to combat MDR bacterial infection.

Z. morio Hemolymph Relieves E. coli-Induced Mastitis by Inhibiting Inflammatory Response and Repairing the Blood-Milk Barrier.

Escherichia coli (E. coli) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio (Z. morio) hemolymph against mastitis pathogens. In this study, we established E. coli-induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli-induced mastitis in vivo and in vitro. In mice with E. coli, Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1ß, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4+ T/CD8+ T, and increased the production of IL-2 triggered by E. coli. Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli-induced mastitis. In E. coli-stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli-induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli-induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli-induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

A Bovine Viral Diarrhea Virus Type 1c Strain in China: Isolation, Identification, and Assessment of Pathogenicity in Rabbits.

Bovine viral diarrhea virus (BVDV) is an important animal pathogen and has a negative economic impact on cattle industries worldwide. In this study, the BVDV strain named BJ175170 was detected, isolated, and identified from cattle in Beijing, China, during herd screening by BVDV antigen-ELISA and indirect immunofluorescence assay (IFA). To investigate its genomic features, the characteristic 5'UTR region of the isolates were sequenced and BLAST analyzed. BVDV BJ175170 belongs to the BVDV-1c subtype, which differs from the Beijing prevalent BVDV strains. The BVDV particles were further observed by transmission electron microscopy (TEM). To evaluate the virulence of the BVDV BJ175170, the BVDV seronegative rabbits were intraperitoneally inoculated with the virus suspension. Blood samples were analyzed for changes in leukocyte number and antibody titer, and tissue samples were taken for histopathology analysis. These data confirmed again that rabbits could act as the reservoir of BVDV, which poses a small but non-zero risk of re-infection for BVDV-free cattle herds. To our knowledge, this is the first report of pathological changes in rabbits after exposure to BVDV-1c subtype, which could act as experimental reference. Meanwhile, the results of this study indicate that rabbits could act as a potential model for studying the mechanism of BVDV in vivo.

Berbamine hydrochloride inhibits bovine viral diarrhea virus replication via interfering in late-stage autophagy.

Bovine viral diarrhea virus (BVDV) is a harmful pathogen that easily causes large-scale infections and huge economic losses to the cattle industry. Berbamine hydrochloride (BBH) is a natural product extracted from berberis and has a wide range of pharmacological effects. However, the antiviral effect of BBH against BVDV needs to be further elucidated. This study aimed to evaluate the antiviral activities of BBH against BVDV infection. We mainly used RT-qPCR, Western blotting, immunofluorescence, and TEM assays to assess the inhibitory activity of BBH against BVDV. The results showed that BBH had an inhibitory effect on BVDV and higher inhibitory activity in the viral attachment and release in MDBK cells. This study found that BVDV could induce and use autophagy to replicate itself. Further results showed that BBH inhibited BVDV infection by inhibiting autophagy integrity in BVDV-infected cells, which was proven by the detection of autophagy-related proteins. Our data show that in BBH-treated BVDV-infected cells, the expression of p62 and LC3 increased over time. After the addition of an autophagy inhibitor, chloroquine (CQ), and an autophagy promoter, rapamycin (Rapa), we found that promoting autophagy was beneficial to the replication of BVDV, while inhibiting autophagy could reduce the number of infections by BVDV, which was evidenced by the expression of the BVDV E2 protein. Furthermore, BBH blocked the normal binding of LC3 and LAMP1 in BVDV-infected cells. In conclusion, BBH inhibited BVDV infection by inhibiting BVDV-induced autophagy in cells, and its inhibitory effect was obvious in the viral attachment and release stages. Therefore, our study provides a new idea for exploring novel anti-BVDV drugs.

A Novel ß-Hairpin Peptide Z-d14CFR Enhances Multidrug-Resistant Bacterial Clearance in a Murine Model of Mastitis.

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH2) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria.

Map of Enteropathogenic Escherichia coli Targets Mitochondria and Triggers DRP-1-Mediated Mitochondrial Fission and Cell Apoptosis in Bovine Mastitis.

Bovine mastitis seriously affects bovine health and dairy product quality. Escherichia coli is the most important pathogen in the environment and dairy products. Enteropathogenic Escherichia coli (EPEC) is a zoonotic pathogen, which seriously threatens the health of people and dairy cows. We recently reported that E. coli can induce endogenous apoptosis in bovine mammary epithelial cells. However, the mechanism of EPEC-damaged mitochondria and -induced bovine mastitis is unclear. In this study, we found that EPEC can induce DRP-1-dependent mitochondrial fission and apoptosis. This was verified by the application of Mdivi, a DRP-1 inhibitor. Meanwhile, in order to verify the role of the Map virulence factor in EPEC-induced bovine mastitis, we constructed a map mutant, complementary strain, and recombinant plasmid MapHis. In the present study, we find that Map induced DRP-1-mediated mitochondrial fission, resulting in mitochondrial dysfunction and apoptosis. These inferences were further verified in vivo by establishing a mouse mastitis model. After the map gene was knocked out, breast inflammation and apoptosis in mice were significantly alleviated. All results show that EPEC targets mitochondria by secreting the Map virulence factor to induce DRP-1-mediated mitochondrial fission, mitochondrial dysfunction, and endogenous apoptosis in bovine mastitis.

Lactobacillus johnsonii L531 Ameliorates Escherichia coli-Induced Cell Damage via Inhibiting NLRP3 Inflammasome Activity and Promoting ATG5/ATG16L1-Mediated Autophagy in Porcine Mammary Epithelial Cells.

Escherichia coli (E. coli), a main mastitis-causing pathogen in sows, leads to mammary tissue damage. Here, we explored the effects of Lactobacillus johnsonii L531 on attenuating E. coli-induced inflammatory damage in porcine mammary epithelial cells (PMECs). L. johnsonii L531 pretreatment reduced E. coli adhesion to PMECs by competitive exclusion and the production of inhibitory factors and decreased E. coli-induced destruction of cellular morphology and ultrastructure. E. coli induced activation of NLRP3 inflammasome associated with increased expression of NLRP3, ASC, and cleaved caspase-1, however, L. johnsonii L531 inhibited E. coli-induced activation of NLRP3 inflammasome. Up-regulation of interleukin (Il)-1ß, Il-6, Il-8, Il-18, tumor necrosis factor alpha, and chemokine Cxcl2 expression after E. coli infection was attenuated by L. johnsonii L531. E. coli infection inhibited autophagy, whereas L. johnsonii L531 reversed the inhibitory effect of E. coli on autophagy by decreasing the expression of autophagic receptor SQSTM1/p62 and increasing the expression of autophagy-related proteins ATG5, ATG16L1, and light chain 3 protein by Western blotting analysis. Our findings suggest that L. johnsonii L531 pretreatment restricts NLRP3 inflammasome activity and induces autophagy through promoting ATG5/ATG16L1-mediated autophagy, thereby protecting against E. coli-induced inflammation and cell damage in PMECs.

Butyrate-mediated autophagy inhibition limits cytosolic Salmonella Infantis replication in the colon of pigs treated with a mixture of Lactobacillus and Bacillus.

Probiotics as an effective and safe strategy for controlling Salmonella infection are much sought after, while autophagy is a central issue in eliminating intracellular pathogens of intestinal epithelial cells. In this study, an animal model of colitis has been developed by infecting weaned pigs orally with a strain of Salmonella Infantis in order to illuminate the potential efficacy of a mixture of Lactobacillus and Bacillus (CBB-MIX) in the resistance to Salmonella infection by regulating butyrate-mediated autophagy. We found that CBB-MIX alleviated S. Infantis-induced colitis and tissue damage. Autophagy markers ATG5, Beclin-1, and the LC3-II/I ratio were significantly enhanced by S. Infantis infection, while treatment with CBB-MIX suppressed S. Infantis-induced autophagy. Additionally, S. Infantis-induced colonic microbial dysbiosis was restored by this treatment, which also preserved the abundance of the butyrate-producing bacteria and the butyrate concentration in the colon. A Caco-2 cell model of S. Infantis infection showed that butyrate had the same effect as the CBB-MIX in restraining S. Infantis-induced autophagy activation. Further, the intracellular S. Infantis load assay indicated that butyrate restricted the replication of cytosolic S. Infantis rather than that in Salmonella-containing vacuoles. Suppression of autophagy by knockdown of ATG5 also attenuated S. Infantis-induced cell injury. Moreover, hyper-replication of cytosolic S. Infantis in Caco-2 cells was significantly decreased when autophagy was inhibited. Our data demonstrated that Salmonella may benefit from autophagy for cytosolic replication and butyrate-mediated autophagy inhibition reduced the intracellular Salmonella load in pigs treated with a probiotic mixture of Lactobacillus and Bacillus.

Antibacterial and immunomodulatory effects of Pheromonicin-NM on Escherichia coli-challenged bovine mammary epithelial cells.

Mastitis affects cows in all regions of the world and Escherichia coli (E. coli) is by far the most common reason of mastitis. Now antibiotic therapy is still the preferred approach of treating mastitis. However, antibiotic usage is easy to lead to antibiotic resistance. There is an urgent need for developing efficacious alternative antimicrobials. Pheromonicin-NM (PMC-NM) is a new engineered bactericidal peptide consisting of colicin Ia and an anti-porin A antibody mimetic. It can lead to the dissipation of cellular energy and therefore kill the bacteria rapidly. The aim of the present study was to investigate the comparative effects of PMC-NM and antibiotic ceftiofur on antibacterial and innate immune responses of bovine mammary epithelial cells (BMEC) to E. coli infection. We found that E. coli growth was inhibited by PMC-NM from 0.5 h after treatment and was completely inhibited at 3 h, indicating a rapid antibacterial activity for PMC-NM. The mRNA expression of TLR2, IL-1ß, IL-8, lactoferrin, LAP, TAP and DEFB1 was increased by PMC-NM treatment at 2 h after E. coli infection, suggesting the enhanced inflammatory responses induced by PMC-NM contribute to pathogens clearance at early phase. By contrast, in E. coli-infected BMECs, ceftiofur treatment upregulated TLR2 and NOD2 levels at 12 h, and extremely elevated transcription levels of TNF-α, IL-1ß, IL-8, lactoferrin, LAP, TAP, BNBD5, DEFB1 at 6 h. The excessive expression of these genes at later phase can induce uncontrolled inflammatory responses and finally cause damage. Taken together, PMC-NM might be used as an ideal antibacterial agent against E. coli mastitis.

An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects.

The regulatory function of Fbxo40 has been well characterized in mice. As a key component of the SCF-E3 ubiquitin ligase complex, Fbxo40 induces IRS1 ubiquitination, thus inactivating the IGF1/Akt pathway. The expression of Fbxo40 is restricted to muscle, and mice with an Fbxo40 null mutation exhibit muscle hypertrophy. However, the function of FBXO40 has not been elucidated in pigs, and it is not known whether FBXO40 mutations affect their health. We therefore generated FBXO40 knockout pigs using somatic cell nuclear transfer (SCNT) technology. CRISPR/Cas9 technology was combined with G418 selection, making it possible to generate donor cells at an efficiency of 75.86%. In muscle from FBXO40 knockout pigs, IRS1 levels were higher, and the IGF1/Akt pathway was stimulated. Mutant animals also had approximately 4% more muscle mass compared to WT controls. The knockout pigs developed normally and no pathological changes were found in major organs. These results demonstrate that FBXO40 is a promising candidate gene for improving production traits in agricultural livestock and for developing therapeutic interventions for muscle diseases.