Expression and characterization of the new antimicrobial peptide AP138L-arg26 anti Staphylococcus aureus.

The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in Pichia pastoris X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G+, minimum inhibitory concentration (MIC) 2-16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill Staphylococcus aureus (S. aureus) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in S. aureus. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future. KEY POINTS: • The AP138L-arg26 was expressed in the P. pastoris expression system with high yield • The AP138 L-arg26 showed high stability and safety in vitro and in vivo • The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism.

Pharmacokinetics and pharmacodynamics of antibacterial peptide NZX in Staphylococcus aureus mastitis mouse model.

Staphylococcus aureus is associated with dairy mastitis, which causes serious economic losses to dairy farming industry. Antibacterial peptide NZX showed good antibacterial activity against S. aureus. This study aimed to evaluate pharmacokinetics and pharmacodynamics of NZX against S. aureus-induced mouse mastitis. NZX exhibited potent in vitro antibacterial activity against the test S. aureus strains (minimal inhibitory concentration (MIC): 0.23-0.46 µM), low mutant prevention concentration (MPC: 1.18-3.68 µM), and a long post antibiotic effect (PAE: 2.20-8.84 h), which was superior to those of lincomycin and ceftiofur. Antibacterial mechanisms showed that NZX could penetrate the cell membrane, resulting in obvious cell membrane perforation and morphological changes, and bind to intracellular DNA. Furthermore, NZX had a good stability in milk environment (retention rate: 85.36%, 24 h) than that in mammary homogenate (47.90%, 24 h). In mouse mastitis model, NZX (25-400 µg/gland) could significantly reduce the bacterial load of mammary tissue in a dose-dependent manner. In addition, NZX (100 µg/gland) could relieve the inflammatory symptoms of mammary tissue, and significantly decreased its pathological scores. The concentration-time curve of NZX (100 µg/gland) in the mammary tissue was plotted and the corresponding pharmacokinetic parameters were obtained by non-compartment model calculation. Those parameters of Tmax, T1/2, Cmax and AUC were 0.5 h, 35.11 h, 32.49 µg/g and 391 µg·h/g, respectively. Therefore, these results suggest that NZX could act as a promising candidate for treating dairy mastitis disease caused by S. aureus. KEY POINTS: • NZX could kill S. aureus by dual mechanism involved in membrane and DNA disruption • NZX could relieve S. aureus-induced mouse mastitis • Pharmacokinetic parameters of NZX in mouse mammary gland were obtained.

Effects of dietary supplementation of bovine lactoferrin on growth performance, immune function and intestinal health in weaning piglets.

Weaning is a crucial period in the pig's life cycle, which is frequently followed by gastrointestinal (GI) infections, diarrhea and even death. This study focused on the impact of bovine lactoferrin (bLF) supplementation on the intestinal health of weaning piglets. Weaning piglets (Duroc × Landrace × Yorkshire, 23 days) were randomly allocated into four groups, which included negative control group (CON): basic diet; positive control group (ANT): basic diet + 20 mg/kg flavomycin + 100 mg/kg aureomycin; treatment group bLF-A: basic diet + 1 g/kg bLF; treatment group bLF-B: basic diet + 3 g/kg bLF. The result showed that dietary supplementation of bLF can improve growth performance and reduce diarrhea, which exhibits dose-dependency (P < 0.05). Compared with CON group, supplementation with bLF significantly improved immunity, and increased villus height and ratio of villus height/crypt depth at the small intestinal mucosa (P < 0.05). The mRNA expression of claudin-1, occludin and ZO-1 was greatly increased in the ileum of bLF group on days 7 and 14 (P < 0.05). Furthermore, the supplementation of bLF increased the abundance of Lactobacillus and Bifidobacterium and decreased the abundance of Escherichia coli in the cecum on day 7 (P < 0.05). The dietary supplementation of bLF enhanced the growth performance, reduced diarrhea rate in weaning piglets by improving intestinal immunity, morphology and barrier function, balancing intestinal microbiota. And bLF can be a promising feed additive in relieving stress situation of weaning piglets.

High-Yield Preparation of American Oyster Defensin (AOD) via a Small and Acidic Fusion Tag and Its Functional Characterization.

The marine peptide, American oyster defensin (AOD), is derived from Crassostrea virginica and exhibits a potent bactericidal effect. However, recombinant preparation has not been achieved due to the high charge and hydrophobicity. Although the traditional fusion tags such as Trx and SUMO shield the effects of target peptides on the host, their large molecular weight (12-20 kDa) leads to the yields lower than 20% of the fusion protein. In this study, a short and acidic fusion tag was employed with a compact structure of only 1 kDa. Following 72 h of induction in a 5 L fermenter, the supernatant exhibited a total protein concentration of 587 mg/L. The recombinant AOD was subsequently purified through affinity chromatography and enterokinase cleavage, resulting in the final yield of 216 mg/L and a purity exceeding 93%. The minimum inhibitory concentrations (MICs) of AOD against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus galactis ranged from 4 to 8 µg/mL. Moreover, time-killing curves indicated that AOD achieved a bactericidal rate of 99.9% against the clinical strain S. epidermidis G-81 within 0.5 h at concentrations of 2× and 4× MIC. Additionally, the activity of AOD was unchanged after treatment with artificial gastric fluid and intestinal fluid for 4 h. Biocompatibility testing demonstrated that AOD, at a concentration of 128 µg/mL, exhibited a hemolysis rate of less than 0.5% and a cell survival rate of over 83%. Furthermore, AOD's in vivo therapeutic efficacy against mouse subcutaneous abscess revealed its capability to restrain bacterial proliferation and reduce bacterial load, surpassing that of antibiotic lincomycin. These findings indicate AOD's potential for clinical usage.

Perspective: A proposal on solutions of modern supply chain construction for lactoferrin.

Lactoferrin is an iron-binding glycoprotein of the transferrin family that is found in most bodily fluids of mammals and has a variety of biological and beneficial functions, with great importance in health enhancement as a supplement for humans and other animals. More than 300 t of lactoferrin were produced in 2021, and this number is expected to grow yearly by 10% to 12%, to over 580 t in 2030. With new and important functions of lactoferrin being revealed and studied, focus on its industrial production and application is increasing accordingly. However, lactoferrin is mainly sourced from cheese whey or skim milk by cation-exchange column chromatography, which is a costly and low-quality method. A potential solution for lactoferrin global supply chain construction is proposed in this article as a complement to traditional routes of purification from whey or skim milk. The large-scale production of lactoferrin, mainly by recombinant yeast, mammal, and grain systems, as well as the market niche and product design, are discussed.

Thinking on the Construction of Antimicrobial Peptide Databases: Powerful Tools for the Molecular Design and Screening.

With the accelerating growth of antimicrobial resistance (AMR), there is an urgent need for new antimicrobial agents with low or no AMR. Antimicrobial peptides (AMPs) have been extensively studied as alternatives to antibiotics (ATAs). Coupled with the new generation of high-throughput technology for AMP mining, the number of derivatives has increased dramatically, but manual running is time-consuming and laborious. Therefore, it is necessary to establish databases that combine computer algorithms to summarize, analyze, and design new AMPs. A number of AMP databases have already been established, such as the Antimicrobial Peptides Database (APD), the Collection of Antimicrobial Peptides (CAMP), the Database of Antimicrobial Activity and Structure of Peptides (DBAASP), and the Database of Antimicrobial Peptides (dbAMPs). These four AMP databases are comprehensive and are widely used. This review aims to cover the construction, evolution, characteristic function, prediction, and design of these four AMP databases. It also offers ideas for the improvement and application of these databases based on merging the various advantages of these four peptide libraries. This review promotes research and development into new AMPs and lays their foundation in the fields of druggability and clinical precision treatment.

C-terminal mini-PEGylation of a marine peptide N6 had potent antibacterial and anti-inflammatory properties against Escherichia coli and Salmonella strains in vitro and in vivo.

BACKGROUND: Enteropathogenic Escherichia coli and Salmonella pullorum are two important groups of zoonotic pathogens. At present, the treatment of intestinal pathogenic bacteria infection mainly relies on antibiotics, which directly inhibit or kill the pathogenic bacteria. However, due to long-term irrational, excessive use or abuse, bacteria have developed different degrees of drug resistance. N6, an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is poorly resistant to enzymatic hydrolysis and distribution in vivo. Polyethylene glycol (PEG) is an extensively studied polymer and commonly used in protein or peptide drugs to improve their therapeutic potential. Here, we modified the N-/C-terminal or Cys residue of N6 with liner PEGn of different lengths (n = 2, 6,12, and 24), and the effects of PEGylation of N6 on the stability, toxicity, bactericidal mechanism, distribution and efficacy were investigated in vitro and in vivo. RESULTS: The antimicrobial activity of the peptide showed that PEGylated N6 at the C-terminus (n = 2, N6-COOH-miniPEG) had potent activity against Gram-negative bacteria; PEGylated N6 at the N-terminus and Cys residues showed low or no activity with increasing lengths of PEG. N6-COOH-miniPEG has higher stability in trypsin than the parent peptide-N6. N6-COOH-miniPEG significantly regulated cytokine expression in lipopolysaccharides (LPS)-induced RAW 264.7 cells, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß were reduced by 31.21%, 65.62% and 44.12%, respectively, lower than those of N6 (-0.06%, -12.36% and -12.73%); N6-COOH-miniPEG increased the level of IL-10 (37.83%), higher than N6 (-10.21%). The data indicated that N6-COOH-miniPEG has more potent anti-inflammatory and immune-regulatory effect than N6 in LPS-stimulated RAW 264.7 cells. N6-COOH-miniPEG exhibited a much wider biodistribution in mice and prolonged in vivo half-time. FITC-labeled N6-COOH-miniPEG was distributed throughout the body of mice in the range of 0.75 - 2 h after injection, while FITC-labeled N6 only concentrated in the abdominal cavity of mice after injection, and the distribution range was narrow. N6-COOH-miniPEG improved the survival rates of mice challenged with E. coli or S. pullorum, downregulated the levels of TNF-α, IL-6, IL-1ß and IL-10 in the serum of LPS-infected mice, and alleviated multiple-organ injuries (the liver, spleen, kidney, and lung), superior to antibiotics, but slightly inferior to N6. CONCLUSIONS: The antibacterial activity, bactericidal mechanism and cytotoxicity of N6-COOH-miniPEG and N6 were similar. N6-COOH-miniPEG has a higher resistance to trysin than N6. The distribution of N6-COOH-miniPEG in mice was superior to that of N6. In exploring the modulatory effects of antimicrobial peptides on cytokines, N6-COOH-miniPEG had stronger anti-inflammatory and immunomodulatory effects than N6. The results suggested that C-terminal PEGylated N6 may provide an opportunity for the development of effective anti-inflammatory and antibacterial peptides.

Antibacterial peptide NZ2114-loaded hydrogel accelerates Staphylococcus aureus-infected wound healing.

Wound infection caused by Staphylococcus aureus (S. aureus) is a great challenge which has caused significant burden and economic loss to the medical system. NZ2114, a plectasin-derived peptide, is an antibacterial agent for preventing and treating S. aureus infection, especially for methicillin-resistant S. aureus (MRSA) infection. Here, three-dimensional reticulated antimicrobial peptide (AMP) NZ2114 hydrogels were developed based on hydroxypropyl cellulose (HPC) and sodium alginate (SA); they displayed sustained and stable release properties (97.88 ± 1.79% and 91.1 ± 10.52% release rate in 72 h, respectively) and good short-term cytocompatibility and hemocompatibility. But the HPC-NZ2114 hydrogel had a smaller pore size (diameter 0.832 ± 0.420 µm vs. 3.912 ± 2.881 µm) and better mechanical properties than that of the SA-NZ2114 hydrogel. HPC/SA-NZ2114 hydrogels possess efficient antimicrobial activity in vitro and in vivo. In a full-thickness skin defect model, the wound closure of the 1.024 mg/g HPC-NZ2114 hydrogel group was superior to those of the SA-NZ2114 hydrogel and antibiotic groups on day 7. The HPC-NZ2114 hydrogel accelerated wound healing by reducing inflammation and promoting the production of vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and angiogenesis (CD31) through histological and immunohistochemistry evaluation. These data indicated that the HPC-NZ2114 hydrogel is an excellent candidate for S. aureus infection wound dressing. KEY POINTS: •NZ2114 hydrogels showed potential in vitro bactericidal activity against S. aureus •NZ2114 hydrogels could release continuously for 72 h and had good biocompatibility •NZ2114 hydrogels could effectively promote S. aureus-infected wound healing.

Resistance response to Arenicin derivatives in Escherichia coli.

The rising prevalence of antibiotic resistance poses the greatest health threats. Antimicrobial peptides (AMPs) are regarded as the potentially effective therapy. To avoid current crisis of antibiotic resistance, a comprehensive understanding of AMP resistance is necessary before clinical application. In this study, the development of resistance to the anti-Gram-negative bacteria peptide N6NH2 (21 residues, ß-sheet) was characterized in E. coli ATCC25922. Three N6NH2-resistant E. coli mutants with 32-fold increase in MIC were isolated by serially passaging bacterial lineages in progressively increasing concentrations of N6NH2 and we mainly focus on the phenotype of N6NH2-resistant bacteria different from sensitive bacteria. The results showed that the resistance mechanism was attributed to synergy effect of multiple mechanisms: (i) increase biofilm formation capacity (3 ~ 4-fold); (ii) weaken the affinity of lipopolysaccharide (LPS) with N6NH2 (3 ~ 8-fold); and (iii) change the cell membrane permeability and potential. Interestingly, a chimeric peptide-G6, also a N6NH2 analog, which keep the same antibacterial activity to both wild-type and resistant clones (MIC value: 16 µg/mL), could curb N6NH2-resistant mutants by stronger inhibition of biofilm formation, stronger affinity with LPS, and stronger membrane permeability and depolarization than that of N6NH2.

A prospective on multiple biological activities of lactoferrin contributing to piglet welfare.

Piglets, especially weaning piglets, show a lower level of immunity and higher morbidity and mortality, owing to their rapid growth, physiological immaturity, and gradual reduction of maternal antibodies, which seriously affects their growth and thus, value. It is important that piglets adapt to nutrient digestion and absorption and develop sound intestinal function and colonization with gut microbiota as soon as possible during their early life stage. Lactoferrin is a natural glycoprotein polypeptide that is part of the transferrin family. It is widely found in mucosal secretions such as saliva and tears, and most highly in milk and colostrum. As a multifunctional bioactive protein and a recommended food additive, lactoferrin is a potential alternative therapy to antibiotics and health promoting additive for piglet nutrition and development. It is expected that lactoferrin, as a natural food additive, could play an important role in maintaining pig health and development. This review examines the following known beneficial effects of lactoferrin: improves the digestion and capacity for absorption in the intestinal tract; promotes the absorption of iron and reduces the incidence of iron deficiency anemia; regulates intestinal function and helps to balance the microbial biota; and enhances the resistance to disease of the piglets via modulating and enhancing the immune system.

A recombinant fungal defensin-like peptide-P2 combats Streptococcus dysgalactiae and biofilms.

Streptococcus dysgalactiae, considered one of the main pathogens that causes bovine mastitis, is a serious threat to humans and animals. However, the excessive use of antibiotics and the characteristic of S. dysgalactiae forming biofilms in mastitic teat canal have serious clinical implications. In this study, in vivo and in vitro multiple mechanisms of action of P2, a mutant of fungal defensin plectasin, against S. dysgalactiae were systematically and comprehensively investigated for the first time. P2 showed potent antibacterial activity against S. dysgalactiae (minimum inhibitory concentration, MIC = 0.23-0.46 µM) and rapid bactericidal action by 3.0 lg units reduction in 2-4 h. No resistant mutants appeared after 30-d serial passage of S. dysgalactiae in the presence of P2. The results of electron microscopy and flow cytometer showed that P2 induced membrane damage of S. dysgalactiae, causing the leakage of cellular content and eventually cell death. Besides, P2 effectively inhibited early biofilm formation, eradicated mature biofilms, and killed 99.9% persisters which were resistant to 100 × MIC vancomycin; and confocal laser scanning microscopy (CLSM) also revealed the potent antibacterial and antibiofilm activity of P2 (the thickness of biofilm reduced from 18.82 to 7.94 µm). The in vivo therapeutic effect of P2 in mouse mastitis model showed that it decreased the number of mammary bacteria and alleviated breast inflammation by regulating cytokines and inhibiting bacterial proliferation, which were superior to vancomycin. These data indicated that P2 maybe a potential candidate peptide for mastitis treatment of S. dysgalactiae infections. KEY POINTS: •P2 showed potential in vitro antibacterial characteristics towards S. dysgalactiae. •P2 eradicated biofilms, killed persisters, and induced cell death of S. dysgalactiae. •P2 could effectively protect mice from S. dysgalactiae infection in gland.

Fatty acid modified-antimicrobial peptide analogues with potent antimicrobial activity and topical therapeutic efficacy against Staphylococcus hyicus.

There is an urgent need to explore new antimicrobial agents due to the looming threat of bacteria resistance. Bovine lactoferricin (LfcinB), as a multifunctional peptide, has the potential to be a new active drug in the future. In this study, it aims to investigate the effect of fatty acid conjugation on antimicrobial peptide activity and topical therapeutic efficacy in a mouse model infected with Staphylococcus hyicus. Both Lfcin4 and Lfcin5 were conjugated with the unsaturated fatty acid linoleic acid (18-C) at their N-terminus and modified by acylation at the C-terminus. The derived peptides of Lin-Lf4NH2 and Lin-Lf5NH2 showed better antibacterial activity (MICs of 3.27 to 6.64 µM) than their parent peptides (MICs of 1.83 to 59.57 µM). Lin-Lf4NH2 (63.2%, 5 min) and Lin-Lf5NH2 (35.8%, 5 min) could more rapidly penetrate bacterial membrane than Lf4NH2 (2.34%, 5 min) and Lf5NH2 (1.94%, 5 min), which further confirmed by the laser scanning confocal microscopy (LSCM). Electron microscopy observations showed Lin-Lf4NH2 and Lin-Lf5NH2 disrupted S. hyicus cell membranes and led to the leakage of contents. Furthermore, after treatment with Lin-Lf4NH2 and Lin-Lf5NH2, the abscess symptoms of mice were significantly alleviated; the recovery rate of abscesses scope of Lin-Lf4NH2 (73.25%) and Lin-Lf5NH2 (71.71%) were 38.8 and 37.9-fold higher than that of untreated group (1.89%), respectively, and superior to Lf4NH2 (46.87%) and Lf5NH2 (58.75%). They significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and chemokine (MCP-1) in S. hyicus skin lesions. This study provides evidence that conjugation of a fatty acid to antimicrobial peptides can improve the activity and have potential for topical therapeutic of S. hyicus skin infections. KEY POINTS: • Lin-Lfcin4NH2/Lfcin5NH2 showed stronger antimicrobial activity than parent peptides. • Lin-Lfcin4NH2/Lfcin5NH2 had a more effective ability to destroy bacterial membranes. • Lin-Lfcin4NH2/Lfcin5NH2 showed a topically higher efficacy than parent peptides.

A study on fungal defensin against multidrug-resistant Clostridium perfringens and its treatment on infected poultry.

In the present study, we aimed to investigate the antibacterial activity and mechanisms of plectasin-derived peptide NZ2114 in vitro and its therapeutic effects in vivo on broilers challenged with Clostridium perfringens. In vitro assay showed that NZ2114 had potent (minimal inhibitory concentration, 0.91 µM) and rapid antibacterial activity (99.9% reduction within 2 h), and the dual antibacterial mechanisms (including interfering with the cell membrane and intracellular DNA) against C. perfringens CVCC 2030. In vivo study, NZ2114 tended to increase linearly and quadratically the average daily gain as NZ2114 level increased and was the highest at 20 mg/L. NZ2114 at 10 ~ 40 mg/L dramatically reduced jejunal lesion score. Besides, the levels of IL-6, TNF-α, and IL-1ß tended to downregulate linearly and quadratically as the NZ2114 level increased and were all the lowest at the dose of 20 mg/L. NZ2114 significantly upregulated those levels of IgA, IgG, IgM, and sIgA with a linear and quadratic dose effect, with the highest IgA, IgG, IgM, and sIgA at 20 mg/L. Finally, NZ2114 tended to linearly and quadratically increase the numerical value of crypt depth, with the lowest value at 40 mg/L. Lincomycin only dramatically reduced the jejunal lesion score and increased the numerical value of crypt depth. These results indicate that NZ2114 has the potential as a new alternative to antibiotics for the treatment of C. perfringens-induced necrotic enteritis infection.Key points• NZ2114 could kill C. perfringens by dual antibacterial mechanisms• Broiler necrotic enteritis model induced by C. perfringens was established• NZ2114 treatment could ameliorate C. perfringens-induced necrotic enteritis.

Potent intracellular antibacterial activity of a marine peptide-N6NH2 and its D-enantiomer against multidrug-resistant Aeromonas veronii.

Aeromonas veronii can cause a variety of diseases such as sepsis in humans and animals. However, there has been no effective way to eradicate A. veronii. In this study, the intracellular antibacterial activities of the C-terminal aminated marine peptide N6 (N6NH2) and its D-enantiomer (DN6NH2) against A. veronii were investigated in macrophages and in mice, respectively. The result showed that DN6NH2 with the minimum inhibitory concentration (MIC) of 1.62 µM is more resistant to cathepsin B than N6NH2 (3.23 µM). The penetration percentages of the cells treated with 4-200 µg/mL fluorescein isothiocyanate (FITC)-DN6NH2 were 52.5-99.6%, higher than those of FITC-N6NH2 (27.0-99.1%). Both N6NH2 and DN6NH2 entered macrophages by macropinocytosis and an energy-dependent manner. DN6NH2 reduced intracellular A. veronii by 34.57%, superior to N6NH2 (19.52%). After treatment with 100 µg/mL DN6NH2, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß were reduced by 53.45%, 58.54%, and 44.62%, respectively, lower than those of N6NH2 (15.65%, 12.88%, and 14.10%, respectively); DN6NH2 increased the IL-10 level (42.94%), higher than N6NH2 (7.67%). In the mice peritonitis model, 5 µmol/kg DN6NH2 reduced intracellular A. veronii colonization by 73.22%, which was superior to N6NH2 (32.45%) or ciprofloxacin (45.67%). This suggests that DN6NH2 may be used as the candidate for treating intracellular multidrug-resistant (MDR) A. veronii. KEY POINTS: • DN6NH2 improved intracellular antibacterial activity against MDR A. veronii. • DN6NH2 entered macrophages by micropinocytosis and enhanced the internalization rates. • DN6NH2 effectively protected the mice from infection with A. veronii.

Design and Pharmacodynamics of Recombinant Fungus Defensin NZL with Improved Activity against Staphylococcus hyicus In Vitro and In Vivo.

Staphylococcus hyicus is recognized as a leading pathogen of exudative epidermitis in modern swine industry. Antimicrobial peptides are attractive candidates for development as potential therapeutics to combat the serious threats of the resistance of S. hyicus. In this study, a series of derivatives were designed based on the NZ2114 template with the aim of obtaining peptides with more potent antimicrobial activity through changing net positive charge or hydrophobicity. Among them, a variant designated as NZL was highly expressed in Pichia pastoris (P. pastoris) with total secreted protein of 1505 mg/L in a 5-L fermenter and exhibited enhanced antimicrobial activity relative to parent peptide NZ2114. Additionally, NZL could kill over 99% of S. hyicus NCTC10350 in vitro within 8 h and in Hacat cells. The results of membrane permeabilization assay, morphological observations, peptide localization assay showed that NZL had potent activity against S. hyicus, which maybe kill S. hyicus through action on the cell wall. NZL also showed an effective therapy in a mouse peritonitis model caused by S. hyicus, superior to NZ2114 or ceftriaxone. Overall, these findings can contribute to explore a novel potential candidate against S. hyicus infections.

A new high-yielding antimicrobial peptide NZX and its antibacterial activity against Staphylococcus hyicus in vitro/vivo.

Staphylococcus hyicus, considered as a leading pathogen of exudative epidermitis, is a serious threat to humans and animals. The emergency of bacterial resistance to antibiotics, especially in human and animal health fields, leads to an urgent need of exploration of new antimicrobial agents. In this study, NZX, a plectasin-derived peptide, was firstly expressed in Pichia pastoris X-33 and was purified by cation exchange chromatography, followed by detection of its antibacterial activity in vitro and in vivo. The results showed that the total secreted protein concentration in fermentation supernatant was up to 2820 mg/L (29 °C) after 120-h induction in a 5-L fermentor. The yield of NZX reached up to 965 mg/L with a purity of 92.6%. The recombinant expressed NZX had a strong antimicrobial activity, high stability, and low toxicity. The minimal inhibitory concentrations (MICs) of NZX and ceftriaxone (CRO) against Gram-positive bacteria were 0.46 to 0.91 µM and 6.04 to 12.09 µM, respectively. The time-killing curves showed that S. hyicus NCTC10350 was killed completely by 2× and 4 × MIC of NZX within 24 h. NZX also exhibited the intracellular activity against S. hyicus in Hacat cells. After treatment with NZX (10 mg/kg) and CRO (60 mg/kg), the survival rates of mice were 100% and 83.3%, respectively. NZX inhibited the bacterial translocation, downregulated pro-inflammatory cytokines (TNF-α/IL-1ß/IL-6), upregulated the anti-inflammatory cytokine (IL-10), and ameliorated multiple-organ injuries (the liver, spleen, lung, and kidney). This study provides evidence that the expressed NZX has the potential to become a powerful candidate as novel antimicrobial agents against S. hyicus infections.

Therapeutic potential of a designed CSαß peptide ID13 in Staphylococcus aureus-induced endometritis of mice.

Staphylococcus aureus is a common pathogen that can cause clinical and subclinical endometritis in humans and animals. In this study, a designed CSαß peptide ID13 from DLP4 exhibited high stable antibacterial activity in simulated gastric fluid (90.79%), serum (99.54%), and different pH buffers (> 99%) against S. aureus CVCC 546 and lower cytotoxicity (89.62% viability) than its parent peptide DLP4 (74.14% viability) toward mouse endometrial epithelial cells (MEECs). ID13 caused a depolarization of bacterial membrane and downregulation of the expression of genes involved in membrane potential maintenance and biofilm formation. The in vitro efficacy analysis of ID13 showed a synergistic effect with vancomycin, ampicillin, rifampin, and ciprofloxacin; intracellular antimicrobial activity against S. aureus CVCC 546 in MEECs; and the ability to inhibit lipoteichoic acid-induced pro-inflammatory cytokines from RAW 264.7. In the S. aureus-induced endometritis of mice, similar to vancomycin, ID13 remarkably alleviated pathological conditions, inhibited the production of cytokines (TNF-α, IL-1ß, IL-6, and IL-10), and suppressed the TLR2-NF-κB signal pathway. Collectively, these results suggest that ID13 could be a potential candidate peptide for therapeutic application in S. aureus-induced endometritis. Key Points •Higher antibacterial activity and lower hemolysis of ID13 than DLP4. •ID13 could downregulate the genes of bacterial survival and infection. •ID13 could alleviate the S. aureus-induced endometritis of mice. •ID13 could regulate the cytokines and suppress the TLR2-NF-κB signal pathway.

Improved Stability and Activity of a Marine Peptide-N6NH2 against Edwardsiella tarda and Its Preliminary Application in Fish.

Edwardsiella tarda can cause fatal gastro-/extraintestinal diseases in fish and humans. Overuse of antibiotics has led to antibiotic resistance and contamination in the environment, which highlights the need to find new antimicrobial agents. In this study, the marine peptide-N6 was amidated at its C-terminus to generate N6NH2. The antibacterial activity of N6 and N6NH2 against E. tarda was evaluated in vitro and in vivo; their stability, toxicity and mode of action were also determined. Minimal inhibitory concentrations (MICs) of N6 and N6NH2 against E. tarda were 1.29-3.2 µM. Both N6 and N6NH2 killed bacteria by destroying the cell membrane of E. tarda and binding to lipopolysaccharide (LPS) and genomic DNA. In contrast with N6, N6NH2 improved the stability toward trypsin, reduced hemolysis (by 0.19% at a concentration of 256 µg/mL) and enhanced the ability to penetrate the bacterial outer and inner membrane. In the model of fish peritonitis caused by E. tarda, superior to norfloxacin, N6NH2 improved the survival rate of fish, reduced the bacterial load on the organs, alleviated the organ injury and regulated the immunity of the liver and kidney. These data suggest that the marine peptide N6NH2 may be a candidate for novel antimicrobial agents against E. tarda infections.

Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii.

Aeromonas veronii is one of the main pathogens causing various diseases in humans and animals. It is currently difficult to eradicate drug-resistant A. veronii due to the biofilm formation by conventional antibiotic treatments. In this study, a marine peptide-N6NH2 and its analogs were generated by introducing Orn or replacing with D-amino acids, Val and Pro; their enzymic stability and antibacterial/antibiofilm ability against multi-drug resistant (MDR) A. veronii ACCC61732 were detected in vitro and in vivo, respectively. The results showed that DN6NH2 more rapidly killed A. veronii ACCC61732 and had higher stability in trypsin, simulated gastric/intestinal fluid, proteinase K, and mouse serum than the parent peptide-N6NH2. DN6NH2 and other analogs significantly improved the ability of N6NH2 to penetrate the outer membrane of A. veronii ACCC61732. DN6NH2, N6PNH2 and V112N6NH2 protected mice from catheter-associated biofilm infection with MDR A. veronii ACCC61732, superior to N6NH2 and CIP. DN6NH2 had more potent efficacy at a dose of 5 µmol/kg (100% survival) in a mouse peritonitis model than other analogs (50-66.67%) and CIP (83.33%), and it inhibited the bacterial translocation, downregulated pro-inflammatory cytokines, upregulated the anti-inflammatory cytokine, and ameliorated multiple-organ injuries (including the liver, spleen, lung, and kidney). These data suggest that the analogs of N6NH2 may be a candidate for novel antimicrobial and antibiofilm agents against MDR A. veronii infections.

A recombinant fungal defensin-like peptide-P2 combats multidrug-resistant Staphylococcus aureus and biofilms.

There is an urgent need to discover new active drugs to combat methicillin-resistant Staphylococcus aureus, which is a serious threat to humans and animals and incompletely eliminated by antibiotics due to its intracellular accumulation in host cells, production of biofilms, and persisters. Fungal defensin-like peptides (DLPs) are emerging as a potential source of new antibacterial drugs due to their potent antibacterial activity. In this study, nine novel fungal DLPs were firstly identified by querying against UniProt databases and expressed in Pichia pastoris, and their antibacterial and anti-biofilm ability were tested against multidrug-resistant (MDR) S. aureus. Results showed that among them, P2, the highest activity and expression level, showed low toxicity, no resistance, and high stability. Minimal inhibitory concentrations (MICs) of P2 against Gram-positive bacteria were < 2 µg/mL. P2 exhibited the potent activity against intracellular MDR S. aureus (bacterial reduction in 80-97%) in RAW264.7 macrophages. P2 bound to/disrupted bacterial DNA, wrinkled outer membranes and permeabilized cytoplasmic membranes, but maintained the integrity of bacterial cells. P2 inhibited/eradicated the biofilm and killed 99% persister bacteria, which were resistant to 100× MIC vancomycin. P2 upregulated the anti-inflammatory cytokine (IL-10) and downregulated pro-inflammatory cytokines (TNF-α/IL-1ß) and chemokine (MCP-1) levels in RAW 264.7 macrophages and in mice, respectively. Five milligram per kilogram P2 enhanced the survival of S. aureus-infected mice (100%), superior to vancomycin (30 mg/kg), inhibited the bacterial translocation, and alleviated multiple-organ injuries (liver, spleen, kidney, and lung). These data suggest that P2 may be a candidate for novel antimicrobial agents against MDR staphylococcal infections.