Molecular characterization and antimicrobial activity of cecropin family in Hermetia illucens.

Antimicrobial peptides are potential alternatives to traditional antibiotics in the face of increasing bacterial resistance. Insects possess many antimicrobial peptides and have become a valuable source of novel and highly effective antimicrobial peptides. Hermetia illucens as a resource insect, for example, has the highest number of antimicrobial peptides of any dipteran. However, most antimicrobial peptides, especially cecropin, have not been comprehensively identified and have not been evaluated for their antimicrobial ability. In this study, we analyzed the localization and gene structure of 33 cecropin molecules in the H. illucens genome and evaluated their activity against common human pathogens. The results showed that 32 cecropin molecules were concentrated on 1 chromosome, most with 2 exons. More importantly, most of the cecropins had a good antibacterial effect against Gram-negative bacteria, and were not hemolytic. The minimum inhibitory concentration (MIC) of the cecropin designated H3 against E. coli was 4 µg/mL. The toxicity, killing time kinetics, and anti-biofilm activity of H3 were further investigated and confirmed its antimicrobial ability. Overall, H3 is a potential candidate for the development of new antimicrobials to treat severe infections caused by Gram-negative pathogens such as E. coli.

Phylogeny of Anopheles darlingi (Diptera:Culicidae) based on the antimicrobial peptide genes cecropin and defensin.

Cecropins and defensins are the main classes of antimicrobial peptides in the mosquito innate immune system, acting against bacteria, fungi and protozoa. There is a knowledge gap concerning these peptide genes in anopheline mosquitoes from the Brazilian Amazon. Thus, this work aimed to describe molecular techniques for detecting the genes encoding the antimicrobial peptides cecropin A (CecA) and defensin in Anopheles darlingi mosquitoes and to perform molecular phylogeny of the sequenced genes using the maximum likelihood method and Bayesian inference with other species from different geographic areas. Our results show, for the first time, a molecular biology method for detecting CecA and defensin in Anopheles darlingi that allows for the use of these molecular markers for phylogenetic analysis in anopheline species, separating the species into single and monophyletic clades.

Novel Antimicrobial Peptides from a Cecropin-Like Region of Heteroscorpine-1 from Heterometrus laoticus Venom with Membrane Disruption Activity.

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

Novel antimicrobial anionic cecropins from the spruce budworm feature a poly-L-aspartic acid C-terminus.

Cecropins form a family of amphipathic α-helical cationic peptides with broad-spectrum antibacterial properties and potent anticancer activity. The emergence of bacteria and cancer cells showing resistance to cationic antimicrobial peptides (CAMPs) has fostered a search for new, more selective and more effective alternatives to CAMPs. With this goal in mind, we looked for cecropin homologs in the genome and transcriptome of the spruce budworm, Choristoneura fumiferana. Not only did we find paralogs of the conventional cationic cecropins (Cfcec+ ), our screening also led to the identification of previously uncharacterized anionic cecropins (Cfcec- ), featuring a poly-l-aspartic acid C-terminus. Comparative peptide analysis indicated that the C-terminal helix of Cfcec- is amphipathic, unlike that of Cfcec+ , which is hydrophobic. Interestingly, molecular dynamics simulations pointed to the lower conformational flexibility of Cfcec- peptides, relative to that of Cfcec+ . Phylogenetic analysis suggests that the evolution of distinct Cfcec+ and Cfcec- peptides may have resulted from an ancient duplication event within the Lepidoptera. Finally, we found that both anionic and cationic cecropins contain a BH3-like motif (G-[KQR]-[HKQNR]-[IV]-[KQR]) that could interact with Bcl-2, a protein involved in apoptosis; this observation is congruent with previous reports indicating that cecropins induce apoptosis. Altogether, our observations suggest that cecropins may provide templates for the development of new anticancer drugs. We also estimated the antibacterial activity of Cfcec-2 and a ∆Cfce-2 peptide as AMPs by testing directly their ability in inhibiting bacterial growth in a disk diffusion assay and their potential for development of novel therapeutics.

Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata.

BACKGROUND: The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages. RESULTS: In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1. CONCLUSIONS: This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

Immune transcriptome analysis in predatory beetles reveals two cecropin genes overexpressed in mandibles.

The great complexity and variety of the innate immune system and the production of antimicrobial peptides in insects is correlated with their evolutionary success and adaptation to different environments. Tiger beetles are an example of non-pest species with a cosmopolitan distribution, but the immune system is barely known and its study could provide useful information about the humoral immunity of predatory insects. Suppression subtractive hybridization (SSH) was performed in Calomera littoralis beetles to obtain a screening of those genes that were overexpressed after an injection with Escherichia coli lipopolysaccharide (LPS). Several genes were identified to be related to immune defense. Among those genes, two members of the cecropin antimicrobial peptides were characterized and identified as CliCec-A and CliCec-B2. Both protein sequences showed cecropin characteristics including 37 and 38 residue mature peptides, composed by two α-helices structures with amphipathic and hydrophobic nature, as shown in their predicted three-dimensional structure. Chemically synthesized CliCec-B2 confirmed cecropin antimicrobial activity against some Gram (+) and Gram (-) bacteria, but not against yeast. Expression of both cecropin genes was assessed by qPCR and showed increases after a LPS injection and highlighted their overexpression in adult beetle mandibles, which could be related to their alimentary habits.

Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants.

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

Antimicrobial functional divergence of the cecropin antibacterial peptide gene family in Musca domestica.

BACKGROUND: It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. METHODS: Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. RESULTS: Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. CONCLUSIONS: Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii.

Design and Unique Expression of a Novel Antibacterial Fusion Protein Cecropin B-Human Lysozyme to Be Toxic to Prokaryotic Host Cells.

A novel antibacterial fusion protein, cecropin B-human lysozyme (CB-hLyso), was designed and expressed in a prokaryotic system. The full-length CB gene was first synthesized and fused to the 5' end of the hLyso gene. The recombinant CB-hLyso was then subcloned in plasmid pET32a, and pET32a-CB-hLyso was transferred into Escherichia coli (E. coli) BL21(DE3) and BL21(DE3)pLysS. The results showed that in the original culture media, Luria-Bertani (LB) media and terrific broth (TB), at 37 or 25 °C, CB-hLyso was barely expressed; however, when the original culture medium was replaced with an equi-volume of fresh medium, obvious expression occurred in BL21(DE3)pLysS/pET32a-CB-hLyso at 25 °C, and the expression in TB (25%) was higher than that in LB (15%). Through a two-step chromatographic method consisting of Ni-chelated Sepharose Fast Flow affinity and Sephadex G-75 size-exclusion, the crude fusion CB-hLyso was isolated in a homogeneous form, and preliminary bacteriostasis experiments showed that the fusion CB-hLyso had a strong inhibitory effect on the growth of Staphylococci. This work provides useful insights into the design of novel fusion polypeptides with higher bacteriolytic activity and wider antimicrobial spectra and in the expression of polypeptide products that are toxic to prokaryotic host cells, eukaryotic host cells or insect cells. Graphical Abstract Schematic representation of expression vector pET-32a-CB-hLyso, with Factor Xa and Asn-Gly.

Expression of a recombinant hybrid antimicrobial peptide magainin II-cecropin B in the mycelium of the medicinal fungus Cordyceps militaris and its validation in mice.

BACKGROUND: Antibiotic residues can cause antibiotic resistance in livestock and their food safety-related issues have increased the consumer demand for products lacking these residues. Hence, developing safe and effective antibiotic alternatives is important to the animal feed industry. With their strong antibacterial actions, antimicrobial peptides have potential as antibiotic alternatives. RESULTS: We investigated the antibacterial and immunomodulatory activities and the mechanisms of action of an antimicrobial peptide. The hybrid antimicrobial peptide magainin II-cecropin B (Mag II-CB) gene was transformed into the medicinal Cordyceps militaris fungus. Recombinant Mag II-CB exhibited broad-spectrum antibacterial activity in vitro and its antibacterial and immunomodulatory functions were evaluated in BALB/c mice infected with Escherichia coli (ATCC 25922). Histologically, Mag II-CB ameliorated E. coli-related intestinal damage and maintained the integrity of the intestinal mucosal barrier by up-regulating tight junction proteins (zonula occludens-1, claudin-1 and occludin). The intestinal microbial flora was positively modulated in the Mag II-CB-treated mice infected with E. coli. Mag II-CB treatment also supported immune functioning in the mice by regulating their plasma immunoglobulin and ileum secreted immunoglobulin A levels, by attenuating their pro-inflammatory cytokine levels, and by elevating their anti-inflammatory cytokines levels. Moreover, directly feeding the infected mice with the C. militaris mycelium producing Mag II-CB further proofed the antibacterial and immunomodulatory functions of recombinant hybrid antimicrobial peptide. CONCLUSION: Our findings suggest that both purified recombinant AMPs and C. militaris mycelium producing AMPs display antibacterial and immunomodulatory activities in mice. And C. militaris producing AMPs has the potential to become a substitute to antibiotics as a feed additive for livestock in future.

Differential Change Patterns of Main Antimicrobial Peptide Genes During Infection of Entomopathogenic Nematodes and Their Symbiotic Bacteria.

The expression of antimicrobial peptides (AMPs) as the main humoral defense reactions of insects during infection by entomopathogenic nematodes (EPNs) and their symbiont is addressed herein. Three AMPs, attacin, cecropin, and spodoptericin, were evaluated in the fifth instar larvae of Spodoptera exigua Hübner (beet armyworm) when challenged with Steinernema carpocapsae or Heterorhabditis bacteriophora. The results indicated that attacin was expressed to a greater extent than either cecropin or spodoptericin. While spodoptericin was expressed to a much lesser extent, this AMP was induced against Gram-positive bacteria, and thus not expressed after penetration of Xenorhabdus nematophila and Photorhabdus luminescens. Attacin and cecropin in the larvae treated with S. carpocapsae at 8 hr post-injection (PI) attained the maximum expression levels and were 138.42-fold and 65.84-fold greater than those of larvae infected with H. bacteriophora, respectively. Generally, the ability of H. bacteriophora to suppress attacin, cecropin, and spodoptericin was greater than that of S. carpocapsae. According to the results, the expression of AMPs by Sp. exigua larvae against S. carpocapsae was determined in the 4 statuses of monoxenic nematode, axenic nematode, live symbiotic bacterium, and dead symbiotic bacterium. The expression of attacin in larvae treated with a monoxenic nematode and live bacterium at 8 and 2 hr PI, respectively, were increased to the maximum amount. Live X. nematophila was the strongest agent for the suppression of attacin. The expression of cecropin against monoxenic nematodes and live symbiotic bacteria at 8 and 4 hr PI, respectively, reached the maximum amount while the expression levels of attacin and cecropin for axenic nematodes were lesser and stable. The results highlighted that the ability of P. luminescens in AMPs suppression was much more than X. nematophila. The results also showed that the effect of symbiotic bacterium in suppressing attacin and cecropin expression was greater than that of a monoxenic nematode; this result provided deep insight into the expression pattern parallels and fluctuations of the main AMPs during nematode infection.

Antimicrobial peptide gene cecropin-2 and defensin respond to peptidoglycan infection in the female adult of oriental fruit fly, Bactrocera dorsalis (Hendel).

Cecropins and defensins are important antimicrobial peptides in insects and are inducible after injection of immune triggers. In this study, we cloned the cDNAs of two antimicrobial peptides (AMPs), cecropin-2 (BdCec-2) and defensin (BdDef) from Bactrocera dorsalis (Hendel), a serious pest causing great economic losses to fruits and vegetables. The BdCec-2 sequence of 192bp encodes a protein of 63 amino acids residues with a predicted molecular weight of 6.78kD. The 282bp cDNA of BdDef encodes a protein of 93 residues with a predicted molecular weight of 9.81kD. Quantitative real-time PCR analyses showed that BdCec-2 and BdDef had similar expression profiles among development stages, the highest mRNA levels of these two AMP genes were observed in the adult stage. Among different adult body segments and tissues, both genes had similar transcriptional profiles, the highest mRNA levels appeared in abdomen and fat body, which was consistent with the reported fact that fat body was the main organ synthesizing AMPs in insects. The expression of BdCec-2 and BdDef were up-regulated after challenge with peptidoglycans from Escherichia coli (PGN-EB) and Staphylococcus aureus (PGN-SA), respectively, suggesting their antimicrobial activity against Gram-negative and Gram-positive microorganisms. These results describe for the first time the basic properties of the cecropin-2 and defensin genes from B. dorsalis that probably play an important role in the defense response against invading microbes.

Characterization and functional study of a Cecropin-like peptide from the Chinese oak silkworm, Antheraea pernyi.

In present study, a Cecropin-like peptide from Antheraea pernyi (ApCec) was cloned and characterized. The full-length ApCec cDNA encoded a protein with 64 amino acids including a putative 22-amino-acid signal peptide, a 4-amino-acid propeptide, and a 38-amino-acid mature peptide. ApCec gene was highly expressed in Malpighian tubules of A. pernyi after induction for 24 h by Escherichia coli in PBS. Pro-ApCec (including propeptide and mature peptide) and M-ApCec (just mature peptide) were synthesized chemically and analyzed by HPLC and mass spectroscopy. The antibacterial activity of M-ApCec is more potent than pro-ApCec against E. coli K12 or B. subtilus in both minimum inhibitory concentration and inhibition zone assays. Hemolytic assay results showed M-ApCec possessed a low cytotoxicity to mammalian cells. The secondary structure of M-ApCec forms α-helical structure, shown by circular dichroism spectroscopy. Transmission electron microscopy analysis suggested that M-ApCec killed bacteria by disrupting bacterial cell membrane integrity. Our results indicate ApCec may play an important role in defending from pathogenic bacteria in A. pernyi, and it may be as a potential candidate for applications in antibacterial drug development and agriculture.

Towards a paradigm shift in innate immunity-seminal work by Hans G. Boman and co-workers.

Four decades ago, immunological research was dominated by the field of lymphoid biology. It was commonly accepted that multicellular eukaryotes defend themselves through phagocytosis. The lack of lymphoid cells in insects and other simpler animals, however, led to the common notion that they might simply lack the capacity defend themselves with humoral factors. This view was challenged by microbiologist Hans G. Boman and co-workers in a series of publications that led to the advent of antimicrobial peptides as a universal arm of the immune system. Besides ingenious research, Boman ignited his work by posing the right questions. He started off by asking himself a simple question: 'Antibodies take weeks to produce while many microbes divide hourly; so how come we stay healthy?'. This led to two key findings in the field: the discovery of an inducible and highly potent antimicrobial immune response in Drosophila in 1972, followed by the characterization of cecropin in 1981. Despite broadly being considered an insect-specific response at first, the work of Boman and co-workers eventually created a bandwagon effect that unravelled various aspects of innate immunity.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Characterization of the Bombyx mori Cecropin A1 promoter regulated by IMD pathway.

Cecropin A1 (CecA1) promoter from Bombyx mori was cloned and characterized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demonstrated that both Escherichia coli and lipopolysaccharide could induce expression in BmE cells but B. bombyseptieus or peptidoglycan failed, and the induction pattern of the reporter gene was coincident with the endogenous CecA1. Analysis of deletion and mutation constructs revealed that the regulatory region was the κB motif located between -176 and -166, and no other predicted elements on CecA1 promoter affected its inducibility. Insertion of additional κB motifs increased the activity of CecA1 promoter. Furthermore, binding of Relish to κB motif was confirmed by electrophoretic mobility shift assay. These findings indicate the regulatory mechanism of CecA1 expression in IMD pathway and suggest an approach of engineering antimicrobial peptide promoter with enhanced activities that may lead to broad applications.

Fusion expression of cecropin B-like antibacterial peptide in Pichia GS115 and its antibacterial mechanism.

OBJECTIVES: To establish an efficient expression system for a fusion protein of glutathione S-transferase and cecropin B (GST-CB) and to clarify the antibacterial mechanism of CB. RESULTS: The optimal incubation time and methanol concentration for induced expression of CB were 36 h and 1 % w/v, respectively. The yield of GST-CB was 2.2 g/l. The minimum inhibitory concentrations of GST-CB towards Staphylococcus aureus subsp. saprophyticus (ATCC 15305) and Escherichia coli strain CFT073 were 250 and 125 µg/ml, respectively. Notably, mutations of proline 24 (P24) in CB produced a polypeptide without antimicrobial activity. CONCLUSION: The fusion protein GST-CB, which has a broad spectrum antimicrobial activity, can be abundantly expressed in Pichia pastoris GS115, and P24 may be an important amino acid for the antimicrobial activity of GST-CB.

A cecropin-like antimicrobial peptide with anti-inflammatory activity from the black fly salivary glands.

BACKGROUND: Several antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, however, the immunological functions for these molecules were poorly understood. METHODS: A novel cecropin-like antimicrobial peptide (SibaCec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaCec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteria Escherichia coli treated with SibaCec were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the mRNA expression of the inducible NO synthase (iNOS) and pro-inflammatory cytokines. Nitric oxide (NO) generation was examined using a Griess assay and the secretion of pro-inflammatory cytokines was determined by an enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase (ERK), p38, and the nuclear translocation of nuclear factor-kappaB (NF-κB) were assessed by Western blotting analysis. Circular dichroism (CD) spectroscopy was performed to evaluate the secondary structure of SibaCec in solvent environment. Interaction of SibaCec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS by SibaCec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression of SibaCec mRNA in the salivary glands of insects after oral infection with the bacteria E.coli. RESULTS: SibaCec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated that SibaCec killed bacteria through the disruption of cell membrane integrity. Furthermore, SibaCec significantly inhibited lipopolysaccharide (LPS)-induced production of NO and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-1ß (IL-1ß) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-κB signaling pathways. It mainly adopted an α-helix conformation in membrane-mimetic environments. SibaCec could interact and neutralize LPS. Infection of black flies with bacteria caused an upregulation of the expression of SibaCec. CONCLUSIONS: These results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.

A potent anti-inflammatory peptide from the salivary glands of horsefly.

BACKGROUND: A diverse group of physiologically active peptides/proteins are present in the salivary glands of horsefly Tabanus yao (Diptera, Tabanidae) that facilitate acquisition of blood meal. However, their roles in the regulation of local inflammation remains poorly understood. METHODS: Induction expression profiles of immune-related molecules in the salivary glands of T. yao was analyzed by quantitative PCR (qPCR) after bacterial feeding. A significantly up-regulated molecule (cecropin-TY1) was selected for anti-inflammatory assay in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The transcription levels of inducible NO synthase (iNOS) and pro-inflammatory cytokines were quantified by qPCR. Nitric oxide (NO) production was determined by Griess reagent. Pro-inflammatory cytokine production was determined by an enzyme-linked immunosorbent assay (ELISA). The inflammatory signals were assayed by Western blotting analysis. The secondary structure of cecropin-TY1 was measured by Circular dichroism (CD) spectroscopy. Interaction of cecropin-TY1 with LPS was evaluated by the dissociation of fluorescein isothiocyanate (FITC)-conjugated LPS aggregates and neutralization of LPS determined by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit. Homology modeled structure analysis and mutation of key residues/structures were performed to understand its structure-activity relationship. RESULTS: Cecropin-TY1 was demonstrated to possess high anti-inflammatory activity and low cytotoxicity toward mouse macrophages. In LPS-stimulated mouse peritoneal macrophage, addition of cecropin-TY1 significantly inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines. Further study revealed that cecropin-TY1 inhibited inflammatory cytokine production by blocking activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signals. Cecropin-TY1 even interacted with LPS and neutralized LPS. The secondary structure analysis revealed that cecropin-TY1 adopted unordered structures in hydrophobic environment but converted to α-helical confirmation in membrane mimetic environments. Homology modeled structure analysis demonstrated that cecropin-TY1 adopted two α-helices (Leu3-Thr24, Ile27-Leu38) linked by a hinge (Leu25-Pro26) and the structure surface was partly positively charged. Structure-activity relationship analysis indicated that several key residues/structures are crucial for its anti-inflammatory activity including α-helices, aromatic residue Trp2, positively charged residues Lys and Arg, hinge residue Pro26 and N-terminal amidation. CONCLUSIONS: We found a novel anti-inflammatory function of horsefly-derived cecropin-TY1 peptide, laying groundwork for better understanding the ectoparasite-host interaction of horsefly with host and highlighting its potency in anti-inflammatory therapy for sepsis and endotoxin shock caused by Gram-negative bacterial infections.

Flow Cytometry and Electron Microscopy Study of Staphylococcus aureus and Escherichia coli Treated with Mdc-Hly.

In our previous study, a novel hybrid protein combining human lysozyme (Hly) with Musca domestica cecropin (Mdc) was successfully constructed. The broad antibacterial activity against various foodborne pathogens of Mdc-hly suggests its scope as a food preservative. The aim of the present study was to investigate the antibacterial mechanism of the recombinant Mdc-hly. The damage induced by Mdc-hly on Staphylococcus aureus and Escherichia coli was investigated using flow cytometry (FC), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results of FC showed that Mdc-hly causes bacterial membrane permeabilization. SEM and TEM studies revealed that Mdc-hly is capable of damaging both the membrane and the wall of bacteria, resulting in efflux of essential cytoplasmic contents. Both FC and EM revealed that the effects of Mdc-hly were greater than its parental peptides. Understanding the antibacterial mechanism of Mdc-hly is of a great interest in further utilization of its use in treatment of food and in clinical environments.

Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli.

Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12h induction with 0.5mM IPTG (isopropyl beta-d-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 µM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 µM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.