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Background Although Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins. Results In this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes. Conclusions The results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents. Graphical Abstract

The skin secretions of many frogs have genetically-encoded, endogenous antimicrobial peptides (AMPs). Other species, especially aposematic poison frogs, secrete exogenously derived alkaloids that serve as potent defense molecules. The origins of these defense systems are not clear, but a novel bile-acid derived metabolite, tauromantellic acid, was recently discovered and shown to be endogenous in poison frogs (Mantella, Dendrobates, and Epipedobates). These observations raise questions about the evolutionary history of AMP genetic elements, the mechanism and function of tauromatellic acid production, and links between these systems. To understand the diversity and expression of AMPs among frogs, we assembled skin transcriptomes of 13 species across the anuran phylogeny. Our analyses revealed a diversity of AMPs and AMP expression levels across the phylogenetic history of frogs, but no observations of AMPs in Mantella. We examined genes expressed in the bile-acid metabolic pathway and found that CYP7A1 (Cytochrome P450), BAAT (bile acid-CoA: amino acid N-acyltransferase), and AMACR (alpha-methylacyl-CoA racemase) were highly expressed in the skin of M. betsileo and either lowly expressed or absent in other frog species. In particular, CYP7A1 catalyzes the first reaction in the cholesterol catabolic pathway and is the rate-limiting step in regulation of bile acid synthesis, suggesting unique activation of the bile acid pathway in Mantella skin. The activation of the bile acid pathway in the skin of Mantella and the lack of observed AMPs fuel new questions about the evolution of defense compounds and the ectopic expression of the bile-acid pathway.

Objectives Anti‐microbial peptides (AMPs) have been comprehensively investigated as a novel alternative to traditional antibiotics against microorganisms. Meanwhile, Tetrahedral DNA nanostructures (TDNs) have gained attention in the field of biomedicine for their premium biological effects and transportation efficiency as delivery vehicles. Hence, in this study, TDN/Histatin 5 (His‐5) was synthesized and the transport efficiency and anti‐fungal effect were measured to evaluate the promotion of His‐5 modified by TDNs. Materials and Methods Tetrahedral DNA nanostructures/His‐5 complex was prepared via electrostatic attraction and characterized by transmission electron microscopy (TEM), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The anti‐fungal effect of the TDN/His‐5 complex was evaluated by determining the growth curve and colony‐forming units of C. albicans. The morphological transformation of C. albicans was observed by light microscope and scanning electron microscope (SEM). Immunofluorescence was performed, and potassium efflux was detected to mechanistically demonstrate the efficacy of TDN/His‐5. Results The results showed that Histatin 5 modified by TDNs had preferable stability in serum and was effectively transported into C. albicans, leading to the increased formation of intracellular reactive oxygen species, higher potassium efflux and enhanced anti‐fungal effect against C. albicans. Conclusions Our study showed that TDN/His‐5 was synthesized successfully. And by the modification of TDNs, His‐5 showed increased transport efficiency and improved anti‐fungal effect., TDN/His‐5 were assembled by TDNs and Histatin 5 by electronic attraction and internalized by Candida albicans, leading to the increased formation of intracellular reactive oxygen species, high potassium efflux, and enhanced antifungal effect against C. albicans.

With rapidly ageing populations, the world is experiencing unsustainable healthcare from chronic diseases such as metabolic, cardiovascular, neurodegenerative, and cancer disorders. Healthy diet and lifestyle might contribute to prevent these diseases and potentially enhance health outcomes in patients during and after therapy. Fermented dairy foods (FDFs) found their origin concurrently with human civilization for increasing milk shelf-life and enhancing sensorial attributes. Although the probiotic concept has been developed more recently, FDFs, such as milks and yoghurt, have been unconsciously associated with health-promoting effects since ancient times. These health benefits rely not only on the occurrence of fermentation-associated live microbes (mainly lactic acid bacteria; LAB), but also on the pro-health molecules (PHMs) mostly derived from microbial conversion of food compounds. Therefore, there is a renaissance of interest toward traditional fermented food as a reservoir of novel microbes producing PHMs, and “hyperfoods” can be tailored to deliver these healthy molecules to humans. In FDFs, the main PHMs are bioactive peptides (BPs) released from milk proteins by microbial proteolysis. BPs display a pattern of biofunctions such as anti-hypertensive, antioxidant, immuno-modulatory, and anti-microbial activities. Here, we summarized the BPs most frequently encountered in dairy food and their biological activities; we reviewed the main studies exploring the potential of dairy microbiota to release BPs; and delineated the main effectors of the proteolytic LAB systems responsible for BPs release.

Within a series of dipeptide derivatives (5&ndash, 11), compound 4 was refluxed with d-glucose, d-xylose, acetylacetone, diethylmalonate, carbon disulfide, ethyl cyanoacetate, and ethyl acetoacetate which yielded 5&ndash, 11, respectively. The candidates 5&ndash, 11 were characterized and their biological activities were evaluated where they showed different anti-microbial inhibitory activities based on the type of pathogenic microorganisms. Moreover, to understand modes of binding, molecular docking was used of Nicotinoylglycine derivatives with the active site of the penicillin-binding protein 3 (PBP3) and sterol 14-alpha demethylase&rsquo, s (CYP51), and the results, which were achieved via covalent and non-covalent docking, were harmonized with the biological activity results. Therefore, it was extrapolated that compounds 4, 7, 8, 9, and 10 had good potential to inhibit sterol 14-alpha demethylase and penicillin-binding protein 3, consequently, these compounds are possibly suitable for the development of a novel antibacterial and antifungal therapeutic drug. In addition, in silico properties of absorption, distribution, metabolism, and excretion (ADME) indicated drug likeness with low to very low oral absorption in most compounds, and undefined blood&ndash, brain barrier permeability in all compounds. Furthermore, toxicity (TOPKAT) prediction showed probability values for all carcinogenicity models were medium to pretty low for all compounds.

Melittin is an anti-microbial peptide (AMP) and one of the most studied membrane-disrupting peptides. There is, however, a lack of accurate measurements of the concentration-dependent kinetics and affinity of binding of melittin to phospholipid membranes. In this study, we used surface plasmon resonance spectroscopy to determine the concentration-dependent effect on the binding of melittin to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers in vesicles. Three concentration ranges were considered, and when combined, covered two orders of magnitudes (0.04 &micro, M to 8 &micro, M), corresponding to concentrations relevant to the membrane-disrupting and anti-microbial activities of melittin. Binding kinetics data were analysed using a 1:1 Langmuir-binding model and a two-state reaction model. Using in-depth quantitative analysis, we characterised the effect of peptide concentration, the addition of NaCl at physiological ionic strength and the choice of kinetic binding model on the reliability of the calculated kinetics and affinity of binding parameters. The apparent binding affinity of melittin for POPC bilayers was observed to decrease with increasing peptide/lipid (P/L) ratio, primarily due to the marked decrease in the association rate. At all concentration ranges, the two-state reaction model provided a better fit to the data and, thus, a more reliable estimate of binding affinity. Addition of NaCl significantly reduced the signal response during the association phase, however, no substantial effect on the binding affinity of melittin to the POPC bilayers was observed. These findings based on POPC bilayers could have important implications for our understanding of the mechanism of action of melittin on more complex model cell membranes of higher physiological relevance.

This study examines the effect of co-administration of antimicrobial peptides and the synthetic glycolipid FP7, which is active in inhibiting inflammatory cytokine production caused by TLR4 activation and signaling. The co-administration of two lipopolysaccharide (LPS)-neutralizing peptides (a cecropin A-melittin hybrid peptide and a human cathelicidin) enhances by an order of magnitude the potency of FP7 in blocking the TLR4 signal. Interestingly, this is not an additional effect of LPS neutralization by peptides, because it also occurs if cells are stimulated by the plant lectin phytohemagglutinin, a non-LPS TLR4 agonist. Our data suggest a dual mechanism of action for the peptides, not exclusively based on LPS binding and neutralization, but also on a direct effect on the LPS-binding proteins of the TLR4 receptor complex. NMR experiments in solution show that peptide addition changes the aggregation state of FP7, promoting the formation of larger micelles. These results suggest a relationship between the aggregation state of lipid A-like ligands and the type and intensity of the TLR4 response. This study was financially supported by the H2020‐MSC‐ETN‐642157 project TOLLerant. The Italian Ministry for Foreign Affairs and International Cooperation (MAECI) is acknowledged. Work at Pompeu Fabra University was supported by MINECO (grants SAF2011‐24899, AGL2014‐52395‐C2‐2‐R to D.A., CTQ2015‐64597‐C2‐1‐P and MINECO‐Severo Ochoa Excellence Acreditation 2017-2021 (SEV‐2016‐0644) to J.J.‐B.) with FEDER funds, and by Generalitat de Catalunya (2014SGR692).

SummaryApart from transporting lipids through the body, the human plasma lipoproteins very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are also thought to serve as a modality for intra-organismal protein transfer, shipping proteins with important roles in inflammation and thrombosis from the site of synthesis to effector locations. To better understand the role of VLDL and LDL in the transport of proteins, we applied a combination of LTQ ORBITRAP-XL (nLC-MS/MS) with both in-SDS-PAGE gel and in-solution tryptic digestion of pure and defined VLDL and LDL fractions. We identified the presence of 95 VLDL-and 51 LDL-associated proteins including all known apolipoproteins and lipid transport proteins, and intriguingly a set of coagulation proteins, complement system and anti-microbial proteins. Prothrombin, protein S, fibrinogen γ, PLTP, CETP, CD14 and LBP were present on VLDL but not on LDL. Prenylcysteine oxidase 1, dermcidin, cathelicidin antimicrobial peptide, TFPI-1 and fibrinogen α chain were associated with both VLDL and LDL. Apo A-V is only present on VLDL and not on LDL. Collectively, this study provides a wealth of knowledge on the protein constituents of the human plasma lipoprotein system and strongly supports the notion that protein shuttling through this system is involved in the regulation of biological processes. Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 – dyslipidaemia, 2 – atherosclerosis and vascular disease, and 3 – coagulation disorders.

Three classes of membrane active peptides (MAPs) are considered in this review: cell penetrating peptides (CPPs); anti-microbial peptides (AMPs), and amyloidal peptides. We summarize both experimental and theoretical results for several representative peptides in these different classes, which highlight commonalities in their interactions with model lipid membranes. While it is clear that no fixed set of mechanisms completely characterize any particular class of MAPs, there is certainly evidence that common mechanisms can be found within and between classes. For example, CPPs appear to undergo rapid translocation across lipid bilayers through small transient pores, which nevertheless appear not to cause persistent damage to membranes. On the other hand, AMPs also show evidence of rapid translocation, but associated with this, is membrane rupture to form large pores, which are subsequently stabilized by peptide adsorption to the pore edges. This disruption to the membrane is presumably responsible for cell death. Amyloidal peptides also show evidence of stable large pore formation, however, the mechanism for pore stabilization appears linked with their ability to form fibrils and prefibrillar aggregates and oligomers. There is some evidence that pores and membrane defects in fact act as nucleation sites for these structures. Where possible we have related the experimental and theoretical work to our own simulation findings in an effort to produce a comprehensive, albeit speculative picture for the mechanisms of action for this important group of peptides.

The human microbiota consists of 100 trillion microorganisms that provide important metabolic and biological functions benefiting the host. However, the presence in host plasma of a gut-derived bacteria component, the lipopolysaccharide (LPS), has been identified as a causal or complicating factor in multiple serious diseases such as sepsis and septic shock and, more recently, obesity-associated metabolic disorders. Understanding the precise mechanisms by which gut-derived LPS is transported from the gut lumen to the systemic circulation is crucial to advance our knowledge of LPS-associated diseases and elaborate targeted strategies for their prevention. The aim of this review is to synthetize current knowledge on the host mechanisms limiting the entry and dissemination of LPS into the systemic circulation. To prevent bacterial colonization and penetration, the intestinal epithelium harbors multiple defense mechanisms including the secretion of antimicrobial peptides and mucins as well as detoxification enzymes. Despite this first line of defense, LPS can reach the apical site of intestinal epithelial cells (IECs) and, because of its large size, likely crosses IECs via transcellular transport, either lipid raft- or clathrin-mediated endocytosis or goblet cell-associated passage. However, the precise pathway remains poorly described. Finally, if LPS crosses the gut mucosa, it is directed via the portal vein to the liver, where major detoxification processes occur by deacetylation and excretion through the bile. If this disposal process is not sufficient, LPS enters the systemic circulation, where it is handled by numerous transport proteins that clear it back to the liver for further excretion.

© 2018 by the authors. This review summarises the current knowledge of Gomesin (Gm), an 18-residue long, cationic anti-microbial peptide originally isolated from the haemocytes of the Brazilian tarantula Acanthoscurria gomesiana. The peptide shows potent cytotoxic activity against clinically relevant microbes including Gram-positive and Gram-negative bacteria, fungi, and parasites. In addition, Gm shows in-vitro and in-vivo anti-cancer activities against several human and murine cancers. The peptide exerts its cytotoxic activity by permeabilising cell membranes, but the underlying molecular mechanism of action is still unclear. Due to its potential as a therapeutic agent, the structure and membrane-binding properties, as well as the leakage and cytotoxic activities of Gm have been studied using a range of techniques. This review provides a summary of these studies, with a particular focus on biophysical characterisation studies of peptide variants that have attempted to establish a structure-activity relationship. Future studies are still needed to rationalise the binding affinity and cell-type-specific selectivity of Gm and its variants, while more pre-clinical studies are required to develop Gm into a therapeutically useful peptide.

The synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified D-phenylalanine residue, their antibacterial properties against several Gram positive and negative strains, as well as their hemolytic activity is reported. (C) 2009 Elsevier Ltd. All rights reserved.

Bacteria produce and excrete a versatile and dynamic suit of compounds to defend against microbial competitors and mediate local population dynamics. These include a wide range of broad-spectrum non-ribosomally synthesized antibiotics, lytic enzymes, metabolic by-products, proteinaceous exotoxins, and ribosomally produced antimicrobial peptides (bacteriocins). Most bacteria produce at least one bacteriocin. Bacteriocins are of interest in the food industry as natural preservatives and in the probiotics industry, leading to extensive studies on lactic acid bacteria (colicin produced by Escherichia coli is a model bacteriocin). Recent studies have projected use of bacteriocins in veterinary medicine and in agriculture, as biostimulants of plant growth and development and as biocontrol agents. For example, bacteriocins such as Cerein 8A, Bac-GM17, putidacin, Bac 14B, amylocyclicin have been studied for their mechanisms of anti-microbial activity. Bac IH7 promotes tomato and musk melon plant growth. Thuricin 17 (Th17) is the only bacteriocin studied extensively for plant growth promotion, including at the molecular level. Th17 functions as a bacterial signal compound, promoting plant growth in legumes and non-legumes. In Arabidopsis thaliana and Glycine max Th17 increased phytohormones IAA and SA at 24 h post treatment. At the proteome level Th17 treatment of 3-week-old A. thaliana rosettes led to >2-fold changes in activation of the carbon and energy metabolism pathway proteins, 24 h post treatment. At 250 mM NaCl stress, the control plants under osmotic-shock shut down most of carbon-metabolism and activated energy-metabolism and antioxidant pathways. Th17 treated plants, at 250 mM NaCl, retained meaningful levels of the light harvesting complex, photosystems I and II proteins and energy and antioxidant pathways were activated, so that rosettes could better withstand the salt stress. In Glycine max, Th17 helped seeds germinate in the presence of NaCl stress, and was most effective at 100 mM NaCl. The 48 h post germination proteome suggested efficient and speedier partitioning of storage proteins, activation of carbon, nitrogen and energy metabolisms in Th17 treated seeds both under optimal and 100 mM NaCl. This review focuses on the bacteriocins produced by plant-rhizosphere colonizers and plant-pathogenic bacteria, that might have uses in agriculture, veterinary, and human medicine.

In Drosophila, anti-microbial peptides are activated and secreted in response to microbial challenge, but the intracellular route of anti-microbial peptide trafficking and the regulatory mechanism controlling their secretion are yet to be fully characterized. We have demonstrated that in Drosophila immune response cells (i.e., fat body cells and hemocytes) the anti-microbial peptide Drosomycin is localized within Rab4 and Rab11 intracellular vesicles. Moreover, both of these small GTPases were required for the delivery of this Drosomycin cargo to the plasma membrane. At the plasma membrane, exocytosis and Drosomycin secretion depend on the SNARE protein Syntaxin1A. Thus, the depletion of Syntaxin1A impaired the release of this antimicrobial peptide, and resulted in the accumulation of Drosomycin and Rab11 carrier vesicles near the plasma membrane. Intriguingly, a similar phenotype was generated by the loss of the adaptor protein 14-3-3ε; there was accumulation of Rab11 vesicles and Drosomycin containing vesicles near the plasma membrane, and a concomitant increase in the susceptibility of 14-3-3ε mutant Drosophila to acute bacterial infection. This suggested that 14-3-3ε, possibly via interaction with Syntaxin1A, is required to promote exocytosis of immune-mediators, thereby regulating innate immune secretion and organism survival under conditions of immune stress. Refereed/Peer-reviewed