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Ability to stimulate antimicrobial immunity has proven to be a useful therapeutic strategy in treating infections, especially in the face of increasing antibiotic resistance. Natural antimicrobial peptides (AMPs) exhibiting immunomodulatory functions normally encompass complex activities, which make it difficult to optimize their therapeutic benefits. Here, a chemotactic motif was harnessed as a template to design a series of AMPs with immunostimulatory activities plus bacteria-killing activities ("AMP plus"). An amphipathic peptide (( Ph HA hP H) n ) was employed to improve the antimicrobial impact and expand the therapeutic potential of the chemotactic motif that lacked obvious bacteria-killing properties. A total of 18 peptides were designed and evaluated for their structure-activity relationships. Among the designed, KWH 2 (1) potently killed bacteria and exhibited a narrow antimicrobial spectrum against Gram-negative bacteria and (2) activated macrophages (i.e., inducing Ca 2+ influx, cell migration, and reactive oxygen species production) as a macrophage chemoattractant. Membrane permeabilization is the major antimicrobial mechanism of KWH 2 . Furthermore, the mouse subcutaneous abscess model supported the dual immunomodulatory and antimicrobial potential of KWH 2 in vivo. The above results confirmed the efficiency of KWH 2 in treating bacterial infection and provided a viable approach to develop immunomodulatory antimicrobial materials with desired properties.

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1 403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1 403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1 403-621 approximately four fold. Moreover, extracellular Ulp1 403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1 403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 10 6 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. KEY POINTS: • First report of extracellular production of Ulp1 403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1 403-621 approximately four fold. • The specific activity of extracellular Ulp1 403-621 produced in this study reached 2.0 × 10 6 U/L., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Due to the alarming developing rate of multidrug-resistant bacterial pathogens, the development and modification of antimicrobial peptides (AMPs) are unprecedentedly active. Despite the fact that considerable efforts have been expended on the discovery and design strategies of AMPs, the clinical translation of peptide antibiotics remains inadequate. A large number of articles and reviews credited the limited success of AMPs to their poor stability in the biological environment, particularly their poor proteolytic stability. In the past forty years, various design strategies have been used to improve the proteolytic stability of AMPs, such as sequence modification, cyclization, peptidomimetics, and nanotechnology. Herein, we focus our discussion on the progress made in improving the proteolytic stability of AMPs and the principle, successes, and limitations of various anti-proteolytic design strategies. It is of prospective significance to extend current insights into the degradation-related inactivation of AMPs and also alleviate/overcome the problem., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Natural antimicrobial peptides have strong bactericidal activities. An obstacle of the development of antimicrobial peptides resides in the difficulty of developing peptides with high biocompatibility. In this study, molecular dynamics analysis was employed to assess the structural characteristics and biological activities of peptides. A (RXKY) 2 (YRY) 2 structure was used as a template to design an antimicrobial peptide RIKL of high-efficiency and low-toxicity, where X represents Ile and Y represents Leu. The secondary structure of the antimicrobial peptide was detected by circular dichroism (CD), and the structures of RIKL in water and in POPC/POPG membrane environment were measured using molecular dynamics. The biological activity of RIKL was further studied by assessing its antimicrobial activity, hemolytic activity, eukaryotic cytotoxicity, and salt ion stability. CD results showed that RIKL presented an α-helical structure in a simulated bacterial membrane environment. Molecular dynamics simulation predicted that the secondary structure of RIKL could be partly retained in water and POPG environment, while this secondary structure was weakened in the POPC environment. Antimicrobial test suggested that RIKL had high antimicrobial activities, and the geometric mean of the Minimum Inhibitory Concentration (MIC) was 3.1 μmol/L. The hemolysis indicated that RIKL had no hemolytic activity within the detection range, and cytotoxicity test suggested the cytotoxicity of RIKL was low. Stability test showed that RIKL maintained antimicrobial activities under different pH, serum concentrations and salt environments. Based on the above results, RIKL has high cell selectivity and has the potential as a highly effective antibacterial drug.

Although antimicrobial peptides (AMPs) have become powerful drug candidates in the post-antibiotic era, but their low protease stability hinders their clinical application. In the present study, the natural sunflower trypsin inhibitor 1 (SFTI-1) binding loop (CTKSIPPIC) was used to design and synthesize a specific anti-proteolytic sequence template ((RX) n W (RX) n CTKSIPPIC (n = 2, 3; X represents A, I, L, V, F, and W)). After several antibacterial, bactericidal, and toxicity tests, RV3 stood out from the variants and had the highest average selectivity index (SI all  = 156.03). It is highly stable in serum, varying pH, temperature, and salt ions as well as under high trypsin, pepsin, or papain concentrations. In a mouse skin inflammation model, established by Pseudomonas aeruginosa infection, RV3 could effectively kill the pathogen, promote wound healing, inhibit inflammatory cell infiltration, and inhibit mRNA and protein expression of TNF-α, IL-6, and IL-1β inflammatory factors. The antibacterial mechanisms of RV3 include combining with lipopolysaccharides and increasing cell membrane permeability, leading to cell membrane rupture and death. These findings indicate that RV3 has great potential for the treatment of bacterial infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Increased antibiotic resistance poses a major limitation in tackling inflammatory bowel disease and presents a large challenge for global health care. Antimicrobial peptides (AMPs) are a potential class of antimicrobial agents. Here, we have designed the potential oral route for antimicrobial peptide R7I with anti‐proteolytic properties to deal with bacterial enteritis in mice. The results revealed that R7I protected the liver and gut from damage caused by inflammation. RNA‐Seq analysis indicated that R7I promoted digestion and absorption in the small intestine by upregulating transmembrane transporter activity, lipid and small molecule metabolic processes and other pathways, in addition to upregulating hepatic steroid biosynthesis and fatty acid degradation. For the gut microbiota, Clostridia were significantly reduced in the R7I‐treated group, and Odoribacteraceae, an efficient isoalloLCA‐synthesizing strain, was the main dominant strain, protecting the gut from potential pathogens. In addition, we further discovered that R7I reduced the accumulation of negative organic acid metabolites. Overall, R7I exerted better therapeutic and immunomodulatory potential in the bacterial enteritis model, greatly reduced the risk of disease onset, and provided a reference for the in vivo application of antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Host defense peptides (HDPs) are small molecules with broad-spectrum antimicrobial activities against infectious bacteria, viruses, and fungi. Increasing evidence suggests that HDPs can also indirectly protect hosts by modulating their immune responses. Due to these dual roles, HDPs have been considered one of the most promising antibiotic substitutes to improve growth performance, intestinal health, and immunity in farm animals. This review describes the antimicrobial and immunomodulatory roles of host defense peptides and their recent applications in animal production. [ABSTRACT FROM AUTHOR]

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1403-621 approximately four fold. Moreover, extracellular Ulp1403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 106 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. Key points: • First report of extracellular production of Ulp1403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1403-621 approximately four fold. • The specific activity of extracellular Ulp1403-621 produced in this study reached 2.0 × 106 U/L. [ABSTRACT FROM AUTHOR]

Treatment of microbial‐associated infections continues to be hampered by impaired antibacterial efficiency and the variability in nanomedicines. Herein, an octapeptide library with a double‐layered zipper, constructed via a systematic arrangement, simplifying the sequence and optimizing the structure (diverse motifs including surfactant‐like, central‐bola, and end‐bola), is assessed in terms of biological efficiency and self‐assembly properties. The results indicate that peptides with double‐layered Trp zipper exhibit significant antimicrobial activity. Extracellularly, affinity interactions between micelles and bacteria induce the lateral flow of the membrane and electric potential perturbation. Intracellularly, lead molecules cause apoptosis‐like death, as indicated by excessive accumulation of reactive oxygen species, generation of a DNA ladder, and upregulation of mazEF expression. Among them, RW‐1 performs the best in vivo and in vitro. The intersecting combination of Trp zipper and surfactants possesses overwhelming superiority with respect to bacterial sweepers (therapeutic index [TI] = 52.89), nanostructures (micelles), and bacterial damage compared to RW‐2 (central‐bola) and RW‐3 (end‐bola). These findings confirm that the combination of double‐layered Trp zipper and surfactants has potential for application as a combined motif for combating microbial infection and connects the vast gap between antimicrobial peptides and self‐assembly, such as Jacob's ladder. [ABSTRACT FROM AUTHOR]

It is traditionally believed that the distribution of tryptophan (Trp) residues is critical for the novo design of antimicrobial peptides (AMPs). However, there is scarce knowledge regarding Trp residues arrangement at the head group level. Thus, a set of α‐helical AMPs containing different Trp residue arrangements at the N‐/C‐terminal of sequence were designed to increase the strategy database and analyze their biological activities. The arrangement of the N‐terminal Trp residue significantly improved the bacteriostatic activity of the peptides, but the C‐terminal Trp residue arrangement reduced the biocompatibility of them. WL and LW were effective against Gram‐negative microbes and had high selectivity for bacteria as compared to human erythrocytes and mammalian cells. They both maintained a relatively desirable activity in the presence of physiological salts and serum. It is observed through electron microscope, flow cytometry and fluorescence spectroscopy that target peptides can penetrate bacterial cell membrane and kill it by damaging cell membrane integrity. Collectively, we determined the structure–activity relationship of Trp residue distributions in a symmetric sequence structure and filled the gap in knowledge related to Trp arrangements at the head group level. The obtained results will be helpful in designing of artificial peptide‐based antimicrobials. [ABSTRACT FROM AUTHOR]

High manufacturing costs and weak cell selectivity have limited the clinical application of naturally occurring peptides when faced with an outbreak of drug resistance. To overcome these limitations, a set of antimicrobial peptides was synthesized with the general sequence of (WL)n, where n = 1, 2, 3, and WL was truncated from the N‐terminus of Cecropin P1 without initial serine residues. The antimicrobial peptide WL3 exhibited stronger antimicrobial activity against both Gram‐negative and Gram‐positive microbes than the parental peptide CP‐1. WL3 showed no hemolysis even at the highest test concentrations compared to the parental peptide CP‐1. The condition sensitivity assays (salts, serum, and trypsin) demonstrated that WL3 had high stability in vitro. Fluorescence spectroscopy and electron microscopy indicated that WL3 killed microbes by means of penetrating the membrane and causing cell lysis. In a mouse model, WL3 was able to significantly reduce the bacteria load in major organs and cytokines (TNF‐α, IL‐6, and IL‐1β) levels in serum. In summary, these findings suggest that WL3, which was modified from a natural antimicrobial peptide, has enormous potential for application as a novel antibacterial agent. [ABSTRACT FROM AUTHOR]

The antimicrobial peptide PMAP-36 exists as a homodimer stabilized by an intermolecular disulfide bridge. The dimer of PMAP-36 exhibits a potent and rapid microbicidal activity against a wide spectrum of microorganisms. The gene encoding the antiparallel dimer (PMAP-36)2 was designed and codon-optimized according to bias of Pichia pastoris. The gene was then expressed in the P. pastoris strain GS115. The concentration of the recombinant product reached 106 mg/l. In vitro activity assays indicated that the recombinant peptide showed antimicrobial activities against Gram-positive and Gram-negative bacteria but did not cause hemolysis of chicken erythrocytes. Subsequently, 120 7-day-old male Arbor Acres broilers were used to evaluate the in vivo activities of the peptide. A prophylactic dose of ciprofloxacin lactate was supplemented as the control. The results showed that recombinant (PMAP-36)2 significantly increased the serum IgM content of the birds (p < 0.05). The recombinant peptide significantly increased the amounts of Bifidobacterium and decreased the amount of Escherichia coli cells in the ceca of the experimental birds (p < 0.05). The results obtained in the present study indicate that the recombinant (PMAP-36)2 has a potent in vitro and in vivo activity and can be used as an alternative to antibiotic treatment. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Antimicrobial peptides (AMPs), which are produced by multicellular organisms as a defense mechanism against competing pathogenic microbes, appear to be excellent candidates for the development of novel antimicrobial agents. Amphipathicity is traditionally believed to be crucial to the de novo design or systematic optimization of AMPs. In this study, we designed a series of short α-helical AMPs with imperfect amphipathicity to augment the arsenal of strategies and to gain further insights into their antimicrobial and hemolytic activity. These imperfectly amphipathic α-helical AMPs were designed by replacing the paired charged amino acid residues on the polar face of an amphipathic peptide with tryptophan residues on the basis of α-helical protein folding principles. PRW4, an imperfectly amphipathic α-helical AMP with hydrogen bonds formed by paired tryptophan residues, was observed to be more selective towards bacterial cells than toward human red blood cells. PRW4 was also effective against Gram-negative and Gram-positive bacteria, and fluorescence spectroscopy, flow cytometry, scanning electron microscopy and transmission electron microscopy indicated that PRW4 killed microbial cells by permeabilizing the cell membrane and damaging their membrane integrity. Therefore, disruptive amphipathicity has excellent potential for the rational design and optimization of AMPs with promising antimicrobial activities. [Copyright &y& Elsevier]

The antimicrobial peptide (AMP) LI is a fusion product of antimicrobial peptide LL37 produced by human neutrophils and Indolicidin secreted by bovine neutrophils. LI retained the antimicrobial activity of the parental peptides and showed high cell selectivity. In this study, the flexible linker Gly-Ser-Gly (G-S-G) was used to ligate LI into dimeric LIG, and constructed the Pichia pastoris (P. pastoris) expression vector pPIC9K-6×His-3×FLAG-LIG. The total protein expression of P. pastoris GS115 reached the highest level (189.6 mg/L) after 96 h induction with 3 % methanol at the initial pH value of 7.0. Finally, 5.9 mg/L of recombinant LIG (rLIG) was obtained after enterokinase digestion and purification. The rLIG had high antimicrobial activity and low hemolytic activity. Compared with monomer LI, GSG linked dimeric LIG, which had no significant change in antimicrobial activity and had good salt ions stability. In this study, the dimeric antimicrobial peptide LIG was successfully expressed, which provided a new idea for the expression of AMPs in the P. pastoris expression system, and had important significance for the application of AMPs. • The flexible linker GSG was used to construct the dimeric antimicrobial peptide LIG. • The antimicrobial peptide LIG was successfully expressed by Pichia pastoris. • The recombinant antimicrobial peptide LIG has high biological activity. [ABSTRACT FROM AUTHOR]

The intestine defends against pathogenic microbial invasion via the secretion of host defense peptides (HDPs). Nutritional immunomodulation can stimulate the expression of endogenous HDPs and enhance the body's immune defense, representing a novel non-antibiotic strategy for disease prevention. The project aims to explore the regulatory mechanism of protegrin-1 (PG-1) expression using sodium phenylbutyrate (PBA) by omics sequencing technology and further investigate the role of key regulatory genes on intestinal health. The results showed that PBA promoted PG-1 expression in intestinal epithelial cells based on cell density through epidermal growth factor receptor (EGFR) and G protein-coupled receptor (GPR43). Transcriptome sequencing and microRNA sequencing revealed that C-X-C motif chemokine receptor 2 (CXCR2) exhibited interactions with PG-1. Pre-treatment cells with a CXCR2 inhibitor (SB225002) effectively suppressed the induction of PG-1 by PBA. Furthermore, SB225002 significantly suppressed the gene expression of HDPs in the jejunum of mice without influencing on the morphology, number of goblet cells, and proliferation of the intestine. CXCR2 inhibition significantly reduced the expression of HDPs during E. coli infection, and resulted in the edema of jejunal epithelial cells. The 16S rDNA analysis of cecal contents showed that the E. coli and SB225002 treatments changed gut microbiota diversity and composition at different taxonomic levels. Correlation analysis suggested a potential regulatory relationship between gut microbiota and HDPs. To that end, a gene involved in the HDP expression, CXCR2, has been identified in the study, which contributes to improving intestinal immune function. PBA may be used as a functional additive to regulate intestinal mucosal function, thereby enhancing the health of the intestinal and host. [ABSTRACT FROM AUTHOR]

Food contamination from microbial deterioration requires the development of potent antimicrobial peptides (AMPs). The deployment of approved AMPs as dietary preservatives is limited due to barriers such as instability, toxicity, and high synthetic costs. This exploration utilizes the primary structural elements of the Trp-pocket backbone to engineer a series of β-hairpin AMPs (XWRWRPGXKXXR-NH 2 , X representing I, V, F, and/or L). Peptides WpLF, with Phe as X and Leu arranged at the 11th position, demonstrated exceptional selectivity index (SI = 123.08) and sterilization effects both in vitro and in vivo. WpLF consistently exhibited stable bacteriostasis, regardless of physiological salts, serum, and extreme pH. Mechanistic analysis indicated that the peptide penetrates microbial cell membranes, inducing membrane disruption, thereby impeding drug resistance evolution. Conclusively, AMPs engineered by the Trp-pocket skeleton hold substantial potential as innovative biological preservatives in food preservation, providing valuable insights for sustainable and safe peptide-based food preservatives. [Display omitted] • Trp-pocket backbone is successfully applied in β-hairpin antimicrobial peptides. • Optimal WpLF exhibits broad-spectrum bacteriostasis and superb biocompatibility. • WpLF targets microbial membrane integrity thus impeding drug resistance evolution. • WpLF proves stable efficacy in physiological barriers and peritonitis-sepsis model. • WpLF offers potential as a food preservative by suppressing bacterial proliferation. [ABSTRACT FROM AUTHOR]

The antimicrobial peptide Cecropin P1 (CP1) was isolated from the pig-intestinal parasitic nematode Ascaris lumbricoides and had potent antimicrobial activity. However, its cytotoxicity is high. Herein, Using CP1 as the parent peptide, we designed a derivative peptide SW by replacing amino acids based on the structure and function relationship of antimicrobial peptides (AMPs) to reduce the toxicity of CP1. Subsequently, the gene sequence encoding SacB-6 ×His-SUMO-SW was constructed into the recombinant expression vector PHY-P43 of Bacillus subtilis WB800N. The fusion proteins with concentrations of 26.97 mg/L were obtained after purification by a Ni–NTA column. A total of 5.37 mg of SW was obtained from 1 L fermentation cultures. Recombinant SW (rSW) exhibits potent antimicrobial activity against gram-negative bacteria by inducing membrane instabilities. In addition, rSW showed low hemolytic activity and cytotoxicity against human red blood cells and IPEC-J2, respectively. In the mice model of peritonitis infection, rSW could reduce Escherichia coli -loading in the liver, spleen, lung, and kidney of mice. These results indicate that rSW production by Bacillus. subtilis is a safe and reliable strategy, and rSW has the potential to be a novel antimicrobial agent against gram-negative bacterial infections. • Low toxic peptide SW derived from Cecropin P1 was expressed in Bacillus subtilis. • The fusion expression strategy and the signal peptide sacB promote heterologous protein secretion. • SW exhibits narrow-spectrum antimicrobial properties and is antimicrobial through a membrane permeabilization mechanism. [ABSTRACT FROM AUTHOR]