Novel design of simplified ß-hairpin antimicrobial peptide as a potential food preservative based on Trp-pocket backbone.

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 ß-hairpin AMPs (XWRWRPGXKXXR-NH2, 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.

Excessive Tryptophan and Phenylalanine Induced Pancreatic Injury and Glycometabolism Disorder in Grower-finisher Pigs.

BACKGROUND: The increase in circulating insulin levels is associated with the onset of type 2 diabetes (T2D), and the levels of branched-chain amino acids and aromatic amino acids (AAAs) are altered in T2D, but whether AAAs play a role in insulin secretion and signaling remains unclear. OBJECTIVES: This study aimed to investigate the effects of different AAAs on pancreatic function and on the use of insulin in finishing pigs. METHODS: A total of 18 healthy finishing pigs (Large White) with average body weight of 100 ± 1.15 kg were randomly allocated to 3 dietary treatments: Con, a normal diet supplemented with 0.68% alanine; Phe, a normal diet supplemented with 1.26% phenylalanine; and Trp, a normal diet supplemented with 0.78% tryptophan. The 3 diets were isonitrogenous. There were 6 replicates in each group. RESULTS: Herein, we investigated the effects of tryptophan and phenylalanine on pancreatic function and the use of insulin in finishing pigs and found that the addition of tryptophan and phenylalanine aggravated pancreatic fat deposition, increased the relative content of saturated fatty acids, especially palmitate (C16:0) and stearate (C18:0), and the resulting lipid toxicity disrupted pancreatic secretory function. We also found that tryptophan and phenylalanine inhibited the growth and secretion of ß-cells, downregulated the gene expression of the PI3K/Akt pathway in the pancreas and liver, and reduced glucose utilization in the liver. CONCLUSIONS: Using fattening pigs as a model, multiorgan combined analysis of the insulin-secreting organ pancreas and the main insulin-acting organ liver, excessive intake of tryptophan and phenylalanine will aggravate pancreatic damage leading to glucose metabolism disorders, providing new evidence for the occurrence and development of T2D.

Dietary pterostilbene exerts potential protective effects by regulating lipid metabolism and enhancing antioxidant capacity on liver in broilers.

Intensive breeding of broilers met the increasing demands of human for broiler products, but it raised their increased susceptibility to various stressors resulting in the disorder of lipid metabolism. Pterostilbene, the methoxylated analogue of resveratrol, exhibits astonishing functions of antioxidant, anti-inflammatory and glycolipid regulatory. The study aimed to elucidate the protective effects of pterostilbene on broiler liver and to explore the potential mechanisms. A total of 480 one-day-old male Arbor Acres (AA) broilers were randomly divided into four groups: the control group (basal diet) and pterostilbene groups (PT200, PT400, and PT600 feeding with basal diet containing 200, 400 and 600 mg/kg pterostilbene, respectively). The results showed that the dietary pterostilbene supplementation significantly improved the ADG of broilers. Dietary pterostilbene supplementation regulated the expression levels of the genes Sirt1 and AMPK and the downstream genes related to lipid metabolism to protect liver function and reduce lipid accumulation in broilers. Dietary pterostilbene supplementation upregulated the expression levels of the Nrf2 gene and its downstream antioxidant genes (SOD, CAT, HO-1, NQO-1, GPX) and phase II detoxification enzyme-related genes (GST, GCLM, GCLC). Collectively, pterostilbene was confirmed the positive effects as a feed additive on lipid metabolism and antioxidant via regulating Sirt1/AMPK and Nrf2 signalling pathways in broilers.

Extreme rainstorm reshuffles the spatial distribution of heavy metals and pollution risk in sediments along the mangrove tidal flat.

Mangroves as typical blue carbon ecosystems exhibit a high level of heavy metal accumulation capability. In this study, we investigated how extreme rainstorm effects the spatial variability and pollution risk of sediment heavy metals (i.e., Fe, Mn, Cr, Cu, Zn, Cd, Pb, As and Hg) at different compartments of a typical tidal flat, including the bare mudflat, mangrove zone, and tidal creek in Shenzhen Bay, China. The results showed that the extreme rainstorm can change the sediment particle size, which further regulated the spatial distribution, and source-sink pattern of heavy metals. Due to the strong rainstorm flushing, the concentrations of most heavy metals increased toward the sea and the comprehensive pollution level increased by 8.3 % after the extreme rainstorm. This study contributes to better understanding of how extreme rainstorm regulates heavy metal behavior in mangrove sediments to achieve sustainable development of mangroves under the pressures of extreme weather events.

Boosting stability and therapeutic potential of proteolysis-resistant antimicrobial peptides by end-tagging ß-naphthylalanine.

Recently, much emphasis has been placed on solving the intrinsic defects of antimicrobial peptides (AMPs), especially their susceptibility to protease digestion for the systemic application of antibacterial biomaterials. Although many strategies have increased the protease stability of AMPs, antimicrobial activity was severely compromised, thereby substantially weakening their therapeutic effect. To address this issue, we introduced hydrophobic group modifications at the N-terminus of proteolysis-resistant AMPs D1 (AArIIlrWrFR) through end-tagging with stretches of natural amino acids (W and I), unnatural amino acid (Nal) and fatty acids. Of these peptides, N1 tagged with a Nal at N-terminus showed the highest selectivity index (GMSI=19.59), with a 6.73-fold improvement over D1. In addition to potent broad-spectrum antimicrobial activity, N1 also exhibited high antimicrobial stability toward salts, serum and proteases in vitro and ideal biocompatibility and therapeutic efficacy in vivo. Furthermore, N1 killed bacteria through multiple mechanisms, involving disruption of bacterial membranes and inhibition of bacterial energy metabolism. Indeed, appropriate terminal hydrophobicity modification opens up new avenues for developing and applying high-stability peptide-based antibacterial biomaterials. STATEMENT OF SIGNIFICANCE: To improve the potency and stability of proteolysis-resistant antimicrobial peptides (AMPs) without increasing toxicity, we constructed a convenient and tunable platform based on different compositions and lengths of hydrophobic end modifications. By tagging an Nal at the N-terminal, the obtained target compound N1 exhibited strong antimicrobial activity and desirable stability under multifarious environments in vitro (proteases, salts and serum), and also showed favorable biocompatibility and therapeutic efficacy in vivo. Notably, N1 exerted its bactericidal effect by damaging bacterial cell membranes and inhibiting bacterial energy metabolism in a dual mode. The findings provide a potential method for designing or optimizing proteolysis-resistant AMPs thus promoting the development and application of peptide-based antibacterial biomaterial.

Characterization of simplified nonapeptides with broad-spectrum antimicrobial activities as potential food preservatives, and their antibacterial mechanism.

Antimicrobial peptides (AMPs) have attracted attention in the field of food preservatives due to their favorable biosafety and potential antimicrobial activity. However, high synthetic cost, systemic toxicity, a narrow antimicrobial spectrum, and poor antimicrobial activity become the main bottlenecks for their practical applications. To address these questions, a set of derived nonapeptides were designed based on a previously discovered ultra-short peptide sequence template (RXRXRXRXL-NH2) and screened to identify an optimal peptide-based food preservative with excellent antimicrobial properties. Among these nonapeptides, the designed peptides 3IW (RIRIRIRWL-NH2) and W2IW (RWRIRIRWL-NH2) presented a membrane-disruptive and reactive oxygen species (ROS) accumulation mechanism to execute potent and rapid broad-spectrum antimicrobial activity without observed cytotoxicity. Moreover, they exhibited favorable antimicrobial stability regardless of high ionic strength, heat, and excessive acid-base conditions, retaining potent antimicrobial effects for chicken meat preservation. Collectively, their ultra-short sequence length and potent broad-spectrum antimicrobial capacity may be beneficial for the further development of green and safe peptide-based food preservatives.

Gut-Derived Metabolites from Dietary Tryptophan Supplementation Quench Intestinal Inflammation through the AMPK-SIRT1-Autophagy Pathway.

Tryptophan has drawn wide attention due to its involvement in improving intestinal immune defense directly and indirectly by regulating metabolic pathways. The study aims to elucidate the potential modulating roles of tryptophan to protect against intestinal inflammation and elucidate the underlying molecular mechanisms. The protective effects of tryptophan against intestinal inflammation are examined in the lipopolysaccharide (LPS)-induced inflammatory model. We first found that tryptophan markedly (p < 0.01) inhibited proinflammatory cytokines production and nuclear factor κB (NF-κB) pathway activation upon LPS challenge. Next, we demonstrated that tryptophan (p < 0.05) attenuated LPS-caused intestinal mucosal barrier damage by increasing the number of goblet cells, mucins, and antimicrobial peptides (AMPs) in the ileum of mice. In addition, tryptophan (p < 0.05) inhibited LPS-induced autophagic flux through the AMP-activated protein kinase (AMPK)-sirtuin 1 (SIRT1) pathway in the intestinal systems to maintain autophagy homeostasis. Meanwhile, tryptophan also reshaped the gut microbiota composition in LPS-challenge mice by increasing the abundance of short-chain fatty acid (SCFA)-producing bacteria such as Acetivibrio (0.053 ± 0.017 to 0.21 ± 0.0041%). Notably, dietary tryptophan resulted in the activation of metabolic pathways during the inflammatory response. Furthermore, exogenous treatment of tryptophan metabolites kynurenine (Kyn) and 5-HT in porcine intestinal epithelial cells (IPEC-J2 cells) reproduced similar protective effects as tryptophan to attenuate LPS-induced intestinal inflammation through regulating the AMPK-SIRT1-autophagy. Taken together, the present study indicates that tryptophan exhibits intestinal protective and immunoregulatory effects resulting from the activation of metabolic pathways, maintenance of gut mucosal barrier integrity, microbiota composition, and AMPK-SIRT1-autophagy level.

Optimized proteolytic resistance motif (DabW)-based U1-2WD: A membrane-induced self-aggregating peptide to trigger bacterial agglutination and death.

The biggest application bottleneck of antimicrobial peptides (AMPs) is the low oral bioavailability caused by the poor stability of digestive enzymes in the gastrointestinal tract. However, the research methods and evaluation criteria of available studies about anti-proteolytic strategies are not uniform and far from the actual environment in vivo. Here, we developed a research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving the protease stability of AMPs on the same platform for the first time. After a comprehensive analysis, Dab modification is identified as the most effective strategy to improve the trypsin stability of AMPs. By further modulating the proteolytic resistance optimization motif (DabW)n, U1-2WD is obtained with ideal stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which forms amorphous aggregates in the bacteria environment to trigger the agglutination of bacterial cells to prevent bacterial escape. It then kills bacteria by disrupting bacterial membranes and inhibiting bacterial energy metabolism. Overall, our work has led to a new understanding of the effectiveness of proteolytic resistance strategies and accelerated the development of anti-proteolytic AMPs to combat multidrug-resistant bacterial infections. STATEMENT OF SIGNIFICANCE: We developed research system and evaluation criteria for proteolytic resistance and systematically evaluated the effectiveness of different strategies for improving protease stability of AMPs on the same platform for the first time. we found effective strategies to resist trypsin hydrolysis: modification with backbone (ß-Arg), D-enantiomer (D-Arg) and L-2,4-diaminobutanoic acid (Dab). Further, the proteolytic resistance optimization motif (DabW)n was designed. When n=3, derivative U1-2WD was obtained with desirable stability and antimicrobial properties in vivo and in vitro. Notably, U1-2WD has a unique antibacterial mechanism, which can self-aggregate into amorphous aggregates in the bacteria environment to mediate the agglutination and sedimentation of bacterial cells to prevent bacterial escape, and then kill bacteria by destroying bacterial membranes and inhibiting bacterial energy metabolism.

Design and heterologous expression of a novel dimeric LL37 variant in Pichia pastoris.

BACKGROUND: The antimicrobial peptide LL37 is produced by white blood cells (mainly neutrophils) and various epithelial cells, and has the outstanding advantages of participating in immune regulation, causing chemotaxis of immune cells and promoting wound healing. However, the central domain of LL37 needs to be improved in terms of antimicrobial activity. RESULTS: In this study, the amino acid substitution method was used to improve the antimicrobial activity of the LL37 active center, and a dimeric design with a better selection index was selected. A flexible linker was selected and combined with the 6 × His-SUMO tag and LG was successfully expressed using Pichia pastoris as a host. Recombinant LG displayed strong antimicrobial activity by destroying the cell membrane of bacteria but had low hemolytic activity. In addition, compared with monomeric peptide FR, rLG had improved ability to tolerate salt ions. CONCLUSION: This research provides new ideas for the production of modified AMPs in microbial systems and their application in industrial production.

Effects of the antimicrobial peptide WK3 on diarrhea, growth performance and intestinal health of weaned piglets challenged with enterotoxigenic Escherichia coli K88.

BACKGROUND: Antibiotics are very effective for treating diarrhea in weaned pigs, but the global prohibition of antibiotics makes it urgent to find an alternative to antibiotics. OBJECTIVE: An experiment was conducted to determine the antimicrobial activity of a linear trpzip-like ß-hairpin antimicrobial peptide WK3 in vivo and to assess its effects on growth performance and intestinal health. DESIGN: Thirty-two piglets were weaned at 21 days and housed in individual metabolic cages, which were randomly divided into four groups and were maintained on a corn-soybean meal-based basal diet. Group 1 included a blank group. Groups 2, 3, and 4 were orally infected by feeding with Enterotoxigenic Escherichia coli (ETEC) K88, which was followed by saline treatment (group 2), enrofloxacin injection at a dose of 2.5 mg/kg (group 3), and WK3 injection at a dose of 2 mg/kg (group 4). The experiment lasted for 6 days, and feed and water were provided ad libitum. RESULTS: Both WK3 and enrofloxacin effectively attenuated diarrhea and improved growth performance of piglets. Compared with the control group, WK3 significantly improved the villus height in the ileum (P < 0.05) but did not affect the villus height in the duodenum or jejunum. Additionally, we did not observe any obvious difference in crypt depth or villus height/crypt depth among the duodenum, jejunum and ileum (P > 0.05). WK3 also reduced the numbers of Enterococcus spp (P < 0.01) in the cecal contents, and the number of Enterobacterium spp tended to decrease (0.05 < P < 0.1). Moreover, the jejunal mucosa of the WK3 group exhibited lower interleukin-1α (IL-1a; P < 0.01), toll-like receptors-4 (TLR-4; P < 0.05), and myeloid differentiation primary response 88 (MyD88; P < 0.01) messenger ribonucleic acid (mRNA) expression levels. The jejunum of the WK3 group also exhibited an increased antioxidant capacity, reduced concentration of malondialdehyde (MDA; P < 0.05), and enhanced superoxide dismutase (SOD) activity (P < 0.05). CONCLUSIONS: WK3 has the potential to replace antibiotics as a new generation feed additive.

A Novel Dual-Targeted α-Helical Peptide With Potent Antifungal Activity Against Fluconazole-Resistant Candida albicans Clinical Isolates.

Due to compromised immune system, fungal infection incidences have markedly increased in the last few decades. Pathogenic fungi have developed resistance to the clinically available antifungal agents. Antifungal resistance poses a great challenge to clinical treatment and has stimulated the demand for novel antifungal agents. A promising alternative to the treatment of fungal diseases is the use of antimicrobial peptides (AMPs). However, the antifungal activities of AMPs have not been fully determined. Therefore, this study aimed at designing and screening α-helical peptides with potential antifungal activities. The effects of key physicochemical parameters on antifungal activities were also investigated. A series of lengthened and residue-substituted derivatives of the template peptide KV, a hexapeptide truncated from the α-helical region of porcine myeloid antimicrobial peptide-36, were designed and synthesized. Enhancement of hydrophobicity by introducing aromatic hydrophobic amino acids (tryptophan and phenylalanine) significantly increased the efficacies of the peptides against Candida albicans strains, including fluconazole-resistant isolates. Increased hydrophobicity also elevated the toxic properties of these peptides. RF3 with moderate hydrophobicity exhibited potent anticandidal activities (GM = 6.96 µM) and modest hemolytic activities (HC10 > 64 µM). Additionally, repeated exposure to a subinhibitory concentration of RF3 did not induce resistance development. The antifungal mechanisms of RF3 were due to membrane disruptions and induction of reactive oxygen species production. Such a dual-targeted mechanism was active against drug-resistant fungi. These results show the important role of hydrophobicity and provide new insights into designing and developing antifungal peptides. Meanwhile, the successful design of RF3 highlights the potential utility of AMPs in preventing the spread of drug-resistant fungal infections.

The optimization of fermentation conditions for Pichia pastoris GS115 producing recombinant xylanase.

Xylanase is a member of an important family of enzymes that has been used in many biotechnological processes. However, the overall cost of enzyme production has been the main problem in the industrial application of enzymes. To obtain maximum xylanase production, statistical approaches based on the Plackett-Burman design and response surface methodology were employed. The results of the statistical analyses demonstrated that the optimal conditions for increased xylanase production were the following: inoculum size, 3.8%; maize meal, 4.5%; histidine, 0.6%; methanol, 1%; culture volume, 20%; bean pulp, 30 g L-1; and Tween-80, 0.8%; and pH 5.0. Verification of the optimization demonstrated that 3273 U mL-1 xylanase was observed under the optimal conditions in shake flask experiments. SDS-PAGE results showed that the size of xylanase protein was about 23 kDa. The results showed that the xylanase produced by fermentation came from Aspergillus Niger by MALDI-TOF-MS. The optimized medium resulted in 2.1- and 1.4-fold higher the activity of xylanase compared with the unoptimized medium (the main nutrients are maize meal and bean pulp) and laboratory medium (the main nutrients are yeast extract and peptone), respectively. The optimization of fermentation conditions is an effective means to reduce production cost and improve xylanase activity.

Maltose Induced Expression of Cecropin AD by SUMO Technology in Bacillus subtilis WB800N.

Cecropin AD (CAD) is a hybrid peptide composed of 37 amino acids with the characters of strong antibacterial, antitumor properties and no hemolytic activity, which was regarded as a promising antibiotic candidate. Thus, a safe method to produce Cecropin AD is necessary to be found. In the study, Bacillus subtilis WB800N was employed as host strain. The CAD coding sequence fused with the signal peptide of SPsacB, the 6 × His gene and the gene of small ubiquitin-like modifier were cloned into the maltose-inducible vector pGJ148. Under the induction by 6% maltose, the recombinant fusion protein was successfully expressed and detected in culture substrate. An optimized amount (26.4 mg/L) of the recombinant CAD was purified of culture supernatant. After purification and digestion, the recombinant CAD was harvested about 4.5 mg/L with a purity of 93%. Recombinant CAD exhibited similar antimicrobial activity with synthetic CAD. This shows that the production of CAD in maltose-induced Bacillus subtilis expression system is a relatively safe method, which is vital for the application of CAD in animal husbandry production.

Heterologous expression of the novel α-helical hybrid peptide PR-FO in Bacillus subtilis.

PR-FO is a novel α-helical hybrid antimicrobial peptide (AMP) with strong antimicrobial activities and high stability, and the potential to develop into a new generation of antimicrobial agents. In this study, the encoded gene sequence of SMT3-PR-FO was designed and transformed into B. subtilis WB800N. Fusion proteins with concentrations of 16 mg L-1 (SPamyQ) and 23 mg L-1 (SPsacB) were obtained after purification by a Ni-NTA resin column. A total of 3 mg (SPamyQ) and 4 mg (SPsacB) of PR-FO with a purity of 90% was obtained from 1 L fermentation cultures. Recombinant PR-FO exhibited high inhibition activities against both gram-negative bacteria and gram-positive bacteria, and low haemolytic activity against human red blood cells. These results indicated that the rSMT3-PR-FO could be expressed under the guidance of SPamyQ and SPsacB, and the maltose-induced expression strategy might be a safe and efficient method for the soluble peptides production in B. subtilis.

A eukaryotic expression strategy for producing the novel antimicrobial peptide PRW4.

The antimicrobial peptide PMAP-36 is a cationic peptide derived from porcine myeloid. The N-terminally paired lysine of PMAP-36 was substituted with tryptophan, and the C-terminal hydrophobic tail was deleted, thereby obtaining the antimicrobial peptide PRW4. PRW4 is a α-helical antimicrobial peptide with broad-spectrum antimicrobial activity. In this study, PRW4 was fused to the 6× His-Trx, and the fusion protein was successfully expressed in Pichia pastoris GS115 from the vector pPICZαA. The maximal induction of recombinant protein occurred in the presence of 1% methanol after 96 h at pH 6.0. After purification by a Ni-NTA resin column and digestion by enterokinase protease, 15 mg of recombinant PRW4 with a purity of 90% was obtained from 1 L of fermentation culture. The results indicated that recombinant PRW4 had similar antimicrobial activity as synthetic PRW4 against bacteria such as Escherichia coli ATCC 25922, Escherichia coli UB 1005, Salmonella typhimurium C7731, Salmonella typhimurium 7913, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, and Streptococcus faecalis ATCC 29212. We have successfully expressed PRW4 in P. pastoris, and this work provides a reference for the production of modified antimicrobial peptides in P. pastoris.

Characterization of an antibacterial dodecapeptide from pig as a potential food preservative and its antibacterial mechanism.

Antimicrobial peptides are believed to be promising bio-preservatives to prevent microbial spoilage through food processing and preservation. With the aim of developing short peptides with broad-spectrum antimicrobial activity and revealing their potential antimicrobial mechanism, a novel class of dodecapeptides were designed by introducing Trp into the hydrophilic face of RI12, a truncated α-helical peptide of porcine myeloid antimicrobial peptide-36 (PMAP-36). The antimicrobial activity study indicated that Trp endowed the peptides with higher antimicrobial potency, and the net charge of +5 was sufficient for the dodecapeptides to exert antimicrobial action. Taking hemolytic activity into consideration, the most promising peptide RI12[K3W] (RLWKIGKVLKWI-NH2) was screened with high antimicrobial activity and non-toxicity. The antimicrobial mechanism study revealed that RI12[K3W] possessed the ability to bind to LPS components and enhance membrane permeability, which was verified by membrane penetration assays. Flow cytometry and electron microscopy further confirmed that RI12[K3W] killed bacterial cells primarily by membrane damage. The results guide the potential application of antimicrobial peptides in the food industry as food preservatives to prevent bacterial contamination.

Therapeutic Potential of Trp-Rich Engineered Amphiphiles by Single Hydrophobic Amino Acid End-Tagging.

End-tagging with a single hydrophobic residue contributes to improve the cell selectivity of antimicrobial peptides (AMPs), but systematic studies have been lacking. Thus, this study aimed to systematically investigate how end-tagging with hydrophobic residues at the C-terminus and Gly capped at the N-terminus of W4 (RWRWWWRWR) affects the bioactivity of W4 variants. Among all the hydrophobic residues, only Ala end-tagging improved the antibacterial activity of W4. Meanwhile, Gly capped at the N-terminus could promote the helical propensity of the end-tagged peptides in dodecylphosphocholine micelles, increasing their antimicrobial activities. Of these peptides, GW4A (GRWRWWWRWRA) showed the best antibacterial activity against the 19 species of bacteria tested (GMMIC = 1.86 µM) with low toxicity, thus possessing the highest cell selectivity (TIall = 137.63). It also had rapid sterilization, good salt and serum resistance, and LPS-neutralizing activity. Antibacterial mechanism studies showed that the short peptide GW4A killed bacteria by destroying cell membrane integrity and causing cytoplasmic leakage. Overall, these findings suggested that systematic studies on terminal modifications promoted the development of peptide design theory and provided a potential method for optimization of effective AMPs.

Rational Design of Short Peptide Variants by Using Kunitzin-RE, an Amphibian-Derived Bioactivity Peptide, for Acquired Potent Broad-Spectrum Antimicrobial and Improved Therapeutic Potential of Commensalism Coinfection of Pathogens.

Commensalism coinfection of pathogens has seriously jeopardized human health. Currently, Kunitzin-RE, as an amphibian-derived bioactivity peptide, is regarded as a potential antimicrobial candidate. However, its antimicrobial properties were unsatisfactory. In this study, a set of shortened variants of Kunitzin-RE was developed by the interception of a peptide fragment and single-site mutation to investigate the effect of chain length, positive charge, hydrophobicity, amphipathicity, and secondary structure on antimicrobial properties. Among them, W8 (AARIILRWRFR) significantly broadened the antimicrobial spectrum and showed the highest antimicrobial activity (GMall = 2.48 µM) against all the fungi and bacteria tested. Additionally, W8 showed high cell selectivity and salt tolerance in vitro, whereas it showed high effectiveness against mice keratitis cause by infection by C. albicans 2.2086. Additionally, it also had obviously lipopolysaccharide-binding ability and a potent membrane-disruptive mechanism. Overall, these findings contributed to the design of short antimicrobial peptides and to combat the serious threat of commensalism coinfection of pathogens.

Antibacterial activities and molecular mechanism of amino-terminal fragments from pig nematode antimicrobial peptide CP-1.

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-1ß) 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.

Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector.

Plectasin, the first fungus defensin, is especially efficient against Gram-positive bacteria. To explore an effective approach for expressing plectasin in Bacillus subtilis, the sequence encoding plectasin fused with the small ubiquitin-like modifier (SUMO) gene, the 6 × His gene and the signal peptide of SacB were cloned into an E. coli-B. subtilis shuttle vector pGJ148 in which the maltose utilization operon promoter Pglv directed the expression. The fusion protein successfully secreted in culture and approximately, 41 mg of the recombinant fusion protein SUMO-plectasin was purified per liter of culture supernatant. After purification by Ni-NTA resin column and digestion by SUMO protease, 5.5 mg of plectasin with a purity of 94 % was obtained from 1 L fermentation culture. Recombinant plectasin was found inhibition activity against S. pneumoniae, S. aureus and S. epidermidis. These results indicate that the maltose-induced expression system may be a safe and efficient way for the large-scale production of soluble peptides in B. subtilis.