The Role of Antimicrobial Peptides in Preterm Birth

Antimicrobial peptides (AMPs) are short cationic amphipathic peptides with a wide range of antimicrobial properties and play an important role in the maintenance of immune homeostasis by modulating immune responses in the reproductive tract. As intra-amniotic infection and microbial dysbiosis emerge as common causes of preterm births (PTBs), a better understanding of the AMPs involved in the development of PTB is essential. The altered expression of AMPs has been reported in PTB-related clinical presentations, such as preterm labor, intra-amniotic infection/inflammation, premature rupture of membranes, and cervical insufficiency. Moreover, it was previously reported that dysregulation of AMPs may affect the pregnancy prognosis. This review aims to describe the expression of AMPs associated with PTBs and to provide new perspectives on the role of AMPs in PTB.     

Aphid BCR4 Structure and Activity Uncover a New Defensin Peptide Superfamily

Aphids (Hemiptera: Aphidoidea) are among the most detrimental insects for agricultural plants, and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR) peptides, provides an alternative to chemical insecticides for pest control. BCRs were initially identified in the pea aphid Acyrthosiphon pisum. They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect cells that host intracellular symbiotic bacteria. Here, we show that one of the A. pisum BCRs, BCR4, displays prominent insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticide. Our comparative genomics and phylogenetic analyses indicate that BCRs are restricted to the aphid lineage. The 3D structure of BCR4 reveals that this peptide belongs to an as-yet-unknown structural class of peptides and defines a new superfamily of defensins.     

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.     

Evaluation of the Binding Mechanism of Human Defensin 5 in a Bacterial Membrane: A Simulation Study

Human α-defensin 5 (HD5) is a host-defense peptide exhibiting broad-spectrum antimicrobial activity. The lipopolysaccharide (LPS) layer on the Gram-negative bacterial membrane acts as a barrier to HD5 insertion. Therefore, the pore formation and binding mechanism remain unclear. Here, the binding mechanisms at five positions along the bacterial membrane axis were investigated using Molecular Dynamics. (MD) simulations. We found that HD5 initially placed at positions 1 to 3 moved up to the surface, while HD5 positioned at 4 and 5 remained within the membrane interacting with the middle and inner leaflet of the membrane, respectively. The arginines were key components for tighter binding with 3-deoxy-d-manno-octulosonic acid (KDO), phosphates of the outer and inner leaflets. KDO appeared to retard the HD5 penetration.     

苍术及其麸炒品抗氧化活性研究

采用清除二苯代苦味酰基(DPPH)自由基、清除[2,2′-连氨-(3-乙基苯并噻唑啉-6-磺酸)]二氨盐(ABTS)自由基和铁离子还原/抗氧化能力(FRAP)测定法对其挥发油和不同提取物进行抗氧化活性的研究。结果表明,生苍术乙酸乙酯提取物具有很好的抗氧化活性(DPPH方法:IC50=6.03μg/mL;ABTS方法:IC50=3.59μg/mL;FRAP方法:FRAP值=3186.67μmolTE/g),麸炒之后,其抗氧化活性显著下降。因而苍术抗氧化应生用。     

Biofilm‐Eradicating Properties of Quaternary Ammonium Amphiphiles: Simple Mimics of Antimicrobial Peptides

Bacterial biofilms are difficult to eradicate because of reduced antibiotic sensitivity and altered metabolic processes; thus, the development of new approaches to biofilm eradication is urgently needed. Antimicrobial peptides (AMPs) and quaternary ammonium cations (QACs) are distinct, yet well‐known, classes of antibacterial compounds. By mapping the general regions of charge and hydrophobicity of QACs onto AMP structures, we designed a small library of QACs to serve as simple AMP mimics. In order to explore the role that cationic charge plays in biofilm eradication, structures were varied with respect to cationic character, distribution of charge, and alkyl side chain. The reported compounds possess minimum biofilm eradication concentrations (MBEC) as low as 25 μM against Gram‐positive biofilms, making them the most active anti‐biofilm structures reported to date. These potent AMP mimics were synthesized in 1–2 steps and hint at the minimal structural requirements for biofilm destruction.     

昆虫抗菌肽的作用机制与应用研究进展

抗菌肽是进化上保守的天然免疫应答成分并且在所有生物体中存在。其抗菌谱宽,对病原性的病毒、细菌、寄生虫和真菌等病原微生物都具有拮抗活性。来源于昆虫的抗菌肽通常是带阳离子的,一般少于100个氨基酸。昆虫抗菌肽的作用机制是通过作用其多样性的靶标来实现的,包括破坏细胞膜、作用细胞质成分和干扰代谢等,但部分昆虫抗菌肽的抗菌机制仍未完全明确,深入了解抗菌肽作用机制将推进昆虫抗菌肽药物开发。对已发现的昆虫抗菌肽的作用机制及其应用进行了综述。     

Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.     

Real-Time Fluorescence Microscopy on Living E. coli Sheds New Light on the Antibacterial Effects of the King Penguin β-Defensin AvBD103b

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich β-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other β-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as β-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a β-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.     

反刍动物内源抗菌肽的应用及发展前景

抗菌肽(microbial peptides,AMPs)又称为抗微生物肽,是在外界因素的诱导作用下,在动物免疫防御系统中生成的一类具有免疫活性的阳离子多肽物质,可抑制细菌、真菌和病毒的生长繁殖,也是防御病原体细菌的首道屏障。反刍动物AMPs可分为防御素及内源性抗菌肽(Cathelicidins),其中Cathelicidins稳定性高,水溶性好,抗菌机理独特,具有广谱抗菌能力,可增强机体免疫力,且不易产生耐药性。本文就反刍动物AMPs的分类、作用机制、生产和转基因的应用及未来发展存在的问题作一综述。     

Lucifensin, a novel insect defensin of medicinal maggots: synthesis and structural study

Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.     

抗菌肽的分子设计研究进展

抗菌肽因其具有广谱高效的抗菌特性,且不易引起耐药性,成为最有潜力的肽类抗生素。为使抗菌肽更加高效、低毒,需对天然抗菌肽进行分子设计改造。就影响抗菌肽抗菌活性的相关因素和分子设计方法等方面的研究进展进行了综述。     

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

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

Aggregation and Its Influence on the Bioactivities of a Novel Antimicrobial Peptide,Temporin-PF,and Its Analogues

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl₂ and MgCl₂ buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.     

Isolation of Peptide Inhibiting SGC-7901 Cell Proliferation from Aspongopus chinensis Dallas

Aspongopus chinensis Dallas is used as a traditional Chinese medicine as well as an edible insect. Although its anti-tumor effects have been observed, the anti-tumor active component(s) in the hemolymph of A. chinensis remains unknown. In this study, a combination usage of ultrafiltration, gel filtration chromatography, FPLC and RP-HPLC to separate and purify active peptides was performed based on the proliferation of the human gastric cancer SGC-7901 cell line treated with candidates. One peptide (MW = 2853.3 Da) was isolated from the hemolymph of A. chinensis. A total of 24 amino acid residues were continuously determined for the active peptide: N′-ECGYCAEKGIRCDDIHCCTGLKKK-C′. In conclusion, a peptide that can inhibit the proliferation of gastric cancer SGC-7901 cells in the hemolymph of A. chinensis was purified in this study, which is homologous to members of the spider toxin protein family. These results should facilitate further works for this peptide, such as the cloning of genes, expression in vitro by prokaryotic or eukaryotic systems, more specific tests of anti-tumor activity, and so on.     

白头翁提取物抗TMV生物活性初探

[目的]探讨白头翁提取物的抗TMV活性,为从植物中开发抗病毒类生物农药提供依据.[方法]以烟草花叶病毒(TMV)为供试病毒,采用半叶枯斑法测定了白头翁提取物对TMV的生物活性.[结果]在供试质量浓度为10 g/L时,白头翁石油醚提取物对TMV具有明显的体外钝化作用,钝化0.5 h后,其抑制率达65.19％,对TMV的初侵染和增殖也有一定的抑制作用,处理24 h后,抑制率分别为46.67％和23.53％.     

Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)–Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-β-xylanases that hydrolyze within the xylan structure, and the β-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.     

Molecular Cloning and Functional Characterization of Tibetan Porcine STING

Tibetan pig is well known for its strong disease resistance. However, little is known about the molecular basis of its strong resistance to disease. Stimulator of interferon (IFN) genes (STING), also known as MPYS/MITA/ERIS/TMEM173, is an adaptor that functions downstream of RIG-I and MAVS and upstream of TBK1 and plays a critical role in type I IFN induction. Here we report the first cloning and characterization of STING gene from Tibetan pig. The entire open reading frame (ORF) of the Tibetan porcine STING is 1137 bp, with a higher degree of sequence similarity with Landrace pig (98%) and cattle (88%) than with chimpanzee (84%), human (83%) or mouse (77%). The predicted protein is composed of 378 amino acids and has 4 putative transmembrane domains. Real-time quantitative PCR analysis indicated that Tibetan pig STING mRNA was most abundant in the lung and heart. Overexpression of Tibetan porcine STING led to upregulation of IFN-β and IFN-stimulated gene 15 (ISG15) in porcine jejunal epithelial cell line IPEC-J2 cells. This is the first study investigating the biological role of STING in intestinal epithelial cells, which lays a foundation for the further study of STING in intestinal innate immunity.