Amphibian-Derived Natural Anticancer Peptides and Proteins: Mechanism of Action, Application Strategies, and Prospects.

Cancer is one of the major diseases that seriously threaten human life. Traditional anticancer therapies have achieved remarkable efficacy but have also some unavoidable side effects. Therefore, more and more research focuses on highly effective and less-toxic anticancer substances of natural origin. Amphibian skin is rich in active substances such as biogenic amines, alkaloids, alcohols, esters, peptides, and proteins, which play a role in various aspects such as anti-inflammatory, immunomodulatory, and anticancer functions, and are one of the critical sources of anticancer substances. Currently, a range of natural anticancer substances are known from various amphibians. This paper aims to review the physicochemical properties, anticancer mechanisms, and potential applications of these peptides and proteins to advance the identification and therapeutic use of natural anticancer agents.

A frog cathelicidin peptide effectively promotes cutaneous wound healing in mice.

Although cathelicidins in mammals have been well characterized, little is known about the function of cathelicidin in amphibians. In the present study, a novel 24-residue peptide (cathelicidin-NV, ARGKKECKDDRCRLLMKRGSFSYV) belonging to the cathelicidin family was identified from the skin of the plateau frog Nanorana ventripunctata Cathelicidin-NV showed strong wound healing-promoting activity in a murine model with a full-thickness dermal wound. It directly enhanced the proliferation of keratinocyte cells, resulting in accelerated re-epithelialization of the wound site. Cathelicidin-NV also promoted the proliferation of fibroblasts, the differentiation of fibroblasts to myofibroblasts and collagen production in fibroblasts, which are implicated in wound contraction and repair processes. Furthermore, cathelicidin-NV promoted the release of monocyte chemoattractant protein-1, tumor necrosis factor-α, vascular endothelial growth factor and transforming growth factor-ß1 in vivo and in vitro, which are essential in the wound-healing processes such as migration, proliferation and differentiation. The MAPK (ERK, JNK and p38) signaling pathways were involved in the wound healing-promoting effect. Additionally, unlike other cathelicidins, cathelicidin-NV did not have any direct effect on microbes and showed no cytotoxicity and hemolytic activity toward mammalian cells at concentrations up to 200 µg/ml. This current study may facilitate the understanding of the cellular and molecular events that underlie quick wound healing in N. ventripunctata In addition, the combination of these properties makes cathelicidin-NV an excellent candidate for skin wound therapeutics.

The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities.

As of February 2017, approximately 7639 amphibian species have been described in the AmphibiaWeb database. However, only 20 cathelicidin-like antimicrobial peptides have been identified to date from 10 amphibian species. Half of these peptides were identified from genome sequences and have not yet been functionally characterized. In this study, a novel cathelicidin-like peptide designated cathelicidin-PP was purified from the skin of tree frog Polypedates puerensis. Cathelicidin-PP is a 32 residue peptide of sequence ASENGKCNLLCLVKKKLRAVGNVIKTVVGKIA. Circular dichroism spectroscopy indicated that cathelicidin-PP mainly adopts a ß-sheet structure in membrane-mimetic solutions. Cathelicidin-PP exhibits potent antimicrobial activity against bacteria and fungi, especially Gram-negative bacteria. Meanwhile, it shows low cytotoxicity toward mammalian cells. Scanning electron microscopy analysis indicated that cathelicidin-PP kills bacteria through the disruption of the bacterial cell membrane integrity. Furthermore, cathelicidin-PP exerts significant anti-inflammatory functions by inhibiting the lipopolysaccharide (LPS)-mediated generation of nitric oxide and pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1ß, and interleukin-6. The MAPKs (ERK, JNK, and p38) and NF-κB signaling pathways are involved in the anti-inflammatory effect. Cathelicidin-PP caused partial neutralization of LPS in a dose-dependent manner. Quantitative PCR indicated that infection of tree frogs with bacteria causes increased expression of cathelicidin-PP in immune-related tissues. Taken together, cathelicidin-PP is the first identified cathelicidin-like peptide from tree frogs. Our findings demonstrate that in addition to direct bactericidal capacity, cathelicidin-PP also possesses immunomodulatory properties, including partial neutralization of LPS, and inhibiting the production of inflammatory cytokines.

A potential wound-healing-promoting peptide from salamander skin.

Although it is well known that wound healing proceeds incredibly quickly in urodele amphibians, such as newts and salamanders, little is known about skin-wound healing, and no bioactive/effector substance that contributes to wound healing has been identified from these animals. As a step toward understanding salamander wound healing and skin regeneration, a potential wound-healing-promoting peptide (tylotoin; KCVRQNNKRVCK) was identified from salamander skin of Tylototriton verrucosus. It shows comparable wound-healing-promoting ability (EC50=11.14 µg/ml) with epidermal growth factor (EGF; NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) in a murine model of full-thickness dermal wound. Tylotoin directly enhances the motility and proliferation of keratinocytes, vascular endothelial cells, and fibroblasts, resulting in accelerated reepithelialization and granulation tissue formation in the wound site. Tylotoin also promotes the release of transforming growth factor ß1 (TGF-ß1) and interleukin 6 (IL-6), which are essential in the wound healing response. Gene-encoded tylotoin secreted in salamander skin is possibly an effector molecule for skin wound healing. This study may facilitate understanding of the cellular and molecular events that underlie quick wound healing in salamanders.

The first anionic defensin from amphibians.

A variety of antimicrobial peptides against infection have been identified from the skin of amphibians. However, knowledge on amphibian defensins is very limited. A novel anionic defensin designated PopuDef was purified from the skin of tree frog Polypedates puerensis, and the cDNA encoding PopuDef precursor was cloned from the skin cDNA library. The amino acid sequence of PopuDef (net charge: -2, pI: 4.75) shared the highest identity of 57 % (25/44) with the salamander defensin CFBD-1 (net charge: 0, pI: 6.14) from urodela amphibians. PopuDef showed moderate antimicrobial activities against P. aeruginosa and S. aureus (MICs are 19.41 and 17.25 µM, respectively), and relatively weak activities against E. coli and B. subtilis (MICs are 38.82 and 43.14 µM, respectively). Tissue distribution analysis indicated that relatively high expression level of PopuDef mRNA was observed in immune-related tissues including skin, gut, lung and spleen. Furthermore, the expression level of PopuDef was significantly upregulated in these tissues after tree frogs were infected with different bacteria strains mentioned above. Interestingly, the induction of PopuDef challenged with E. coli or B. subtilis, which was less sensitive to PopuDef, was much higher than that did with P. aeruginosa or S. aureus. These findings highlight the key role of PopuDef in innate immunity against infection. To our knowledge, PopuDef is the first anionic defensin characterized from amphibians.

An amphibian-derived cathelicidin accelerates cutaneous wound healing through its main regulatory effect on phagocytes.

Cathelicidins are an important family of antimicrobial peptides (AMPs) involved in the innate immunity in vertebrates. The mammalian cathelicidins have been well characterized, but the relationship between structure and function in amphibian cathelicidins is still not well understood. In this study, a novel 29-residue cathelicidin antimicrobial peptide (BugaCATH) was identified from the skin of Bufo gargarizans. Unlike other AMPs, BugaCATH does not display any direct antimicrobial effects in vitro. However, it effectively promotes full-thickness wound repair in mice. Following injury, BugaCATH initiates and expedites the inflammatory stage by recruiting neutrophils and macrophages to the wound site. BugaCATH not only regulates neutrophil phagocytic activity but also stimulates the generation of cytokines (TNF-α, IL-6, and IL-1ß) and chemokines (CXCL1, CXCL2, CCL2, and CCL3) in macrophages and in mice. Furthermore, it promotes macrophage M2 polarization that facilitates the conversion from a pro-inflammatory macrophage-dominated wound environment to an anti-inflammatory one during the mid to late stages, which is crucial for reducing inflammation and effective wound repair. The MAPK (ERK, JNK, and p38) and NF-κB-NLRP3 signaling pathways are involved in the activity. Moreover, BugaCATH directly enhances the migration of keratinocytes and vascular endothelial cells without affecting their proliferation. Notably, BugaCATH significantly improves the proliferation of keratinocytes and endothelial cells in the presence of macrophages. The current study revealed that in addition to proliferation of keratinocytes and endothelial cells, BugaCATH possesses the ability to modulate inflammatory processes during skin injury through its regulatory effect on phagocytes. The combination of these capabilities makes BugaCATH a potent candidate for skin wound therapy.

A non-bactericidal cathelicidin with antioxidant properties ameliorates UVB-induced mouse skin photoaging via intracellular ROS scavenging and Keap1/Nrf2 pathway activation.

Cathelicidins, a category of critical host defense molecules in vertebrates, have been extensively studied for their bactericidal functions, but little is known about their non-bactericidal properties. Herein, a novel cathelicidin peptide (Atonp2) was identified from the plateau frog Nanorana ventripunctata. It did not exhibit bactericidal activity but showed significant therapeutic effects in chronic UVB radiation-induced mouse skin photoaging through inhibiting thickening, pyroptosis and inflammation in the epidermis, while inhibiting cellular senescence, collagen fibre breakage and type Ⅰ collagen reduction in the dermis. Further studies indicated that Atonp2 effectively scavenged UVB-induced intracellular ROS via tyrosines at positions 9 and 10, while activating the Keap1/Nrf2 pathway to protect epidermal keratinocytes against UVB radiation, which in turn indirectly reversed the senescence and collagen degradation of dermal fibroblasts, thereby ameliorating UVB-induced skin photoaging. As such, this study identified a non-bactericidal cathelicidin peptide with potent antioxidant functions, highlighting its potential to treat and prevent skin photoaging.

A Nonbactericidal Anionic Antimicrobial Peptide Provides Prophylactic and Therapeutic Efficacies against Bacterial Infections in Mice by Immunomodulatory-Antithrombotic Duality.

Although bactericidal cationic antimicrobial peptides (AMPs) have been well characterized, less information is available about the antibacterial properties and mechanisms of action of nonbactericidal AMPs, especially nonbactericidal anionic AMPs. Herein, a novel anionic antimicrobial peptide (Gy-CATH) with a net charge of -4 was identified from the skin of the frog Glyphoglossus yunnanensis. Gy-CATH lacks direct antibacterial effects but exhibits significantly preventive and therapeutic capacities in mice that are infected with Staphylococcus aureus, Enterobacteriaceae coli, methicillin-resistant Staphylococcus aureus (MRSA), or carbapenem-resistant E. coli (CREC). In vitro and in vivo investigations proved the regulation of Gy-CATH on neutrophils and macrophages involved in the host immune defense against infection. Moreover, Gy-CATH significantly reduced the extent of pulmonary fibrin deposition and prevented thrombosis in mice, which was attributed to the regulatory role of Gy-CATH in physiological anticoagulants and platelet aggregation. These findings show that Gy-CATH is a potential candidate for the treatment of bacterial infection.

Natural Amphibian-Derived Host Defense Peptides: Peptide Immunomodulators with Potential Therapeutic Value.

Due to the rapid evolution of bacterial drug resistance, anti-infective treatment has become a global problem. Therefore, there is an urgent need to develop alternative treatment strategies. Host defense peptides (HDPs) are important components of the natural immune system and are widely distributed in the animal and plant kingdoms. Amphibians, especially their skin, provide a rich source of natural HDPs encoded by genes. These HDPs exhibit not only broad-spectrum antimicrobial activity but also a wide range of immunoregulatory characteristics, including modulation of antiinflammatory and proinflammatory reactions, regulation of specific cellular functions, enhancement of immune chemotaxis, regulation of adaptive immunity, and promotion of wound healing. They also show potent therapeutic effects on infectious and inflammatory diseases caused by pathogenic microorganisms. Thus, in the current review, we summarize the extensive immunomodulatory functions of natural amphibian HDPs, as well as the challenges of clinical development and potential solutions, which have important implications for the development of new anti-infective drugs.

Tree Frog-Derived Cathelicidin Protects Mice against Bacterial Infection through Its Antimicrobial and Anti-Inflammatory Activities and Regulatory Effect on Phagocytes.

Due to excessive use or abuse in the food industry, agriculture, and medicine, many pathogens are developing resistance against conventional antibiotics. Antimicrobial peptides (AMPs) hold promise as effective therapeutic options for the treatment of bacterial infections. Herein, a novel cathelicidin antimicrobial peptide (Zs-CATH) was identified from the tree frog Zhangixalus smaragdinus. Zs-CATH mainly adopted an amphipathic ß-sheet structure in a membrane-mimetic environment. It showed broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria in vitro and significantly protected mice from lethal infections induced by Gram-negative bacteria Escherichia coli ATCC 25922 or Gram-positive bacteria Staphylococcus aureus ATCC 25923 in vivo. In addition, Zs-CATH exerted a strong anti-inflammatory effect by neutralizing lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and promoting macrophage M2 polarization, thus inhibiting the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) and enhancing the production of M2 macrophage markers IL-10, IL-4, and CD206. The MAPK and NF-κB inflammatory signaling pathways and transcriptional activator 6 (STAT6) were involved in this effect. In mice, Zs-CATH rapidly recruited neutrophils and monocytes/macrophages to the abdominal cavity but not T and B lymphocytes. Zs-CATH did not exhibit a direct chemoattractant effect on phagocytes but significantly promoted phagocyte migration in the presence of macrophages. Zs-CATH stimulated macrophages to secrete chemokines CXCL1, CXCL2, and CCL2, which mediated the recruitment of phagocytes. Furthermore, Zs-CATH promoted the production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs), which are oxygen-dependent and oxygen-independent mechanisms of the microbicidal activity of neutrophils, respectively. Zs-CATH exhibited no toxic side effects on mammalian cells and mice. These findings show that in addition to direct antibacterial activity, Zs-CATH also possesses the ability to modulate immune and inflammatory processes during bacterial infection, showing potential for development as anti-infective and/or anti-inflammatory agents.

A Frog-Derived Cathelicidin Peptide with Dual Antimicrobial and Immunomodulatory Activities Effectively Ameliorates Staphylococcus aureus-Induced Peritonitis in Mice.

As antimicrobial resistance poses an increasing threat to public health, it is urgent to develop new antimicrobial agents. In this paper, we identify a novel 30-residue peptide (Nv-CATH, NCNFLCKVKQRLRSVSSTSHIGMAIPRPRG) from the skin of the frog Nanorana ventripunctata, which belongs to the cathelicidin family. Nv-CATH exhibited broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. Nv-CATH significantly protected mice from lethal infections caused by Staphylococcus aureus. Furthermore, the peptide suppressed excessive and harmful inflammatory responses by repressing the production of NO, IL-6, TNF-α, and IL-1ß. The NF-κB-NLRP3 and MAPK inflammatory signaling pathways were involved in the protection in vitro and in vivo. Nv-CATH also modulated macrophage/monocyte and neutrophil trafficking to the infection site by stimulating CXCL1, CXCL2, and CCL2 production in macrophages. Nv-CATH augmented immunocyte-mediated bacterial killing by modestly promoting neutrophils' phagocytosis and PMA-induced NET formation. Thus, Nv-CATH protects mice against bacterial infection by antimicrobial-immunomodulatory duality. The combination of these two characteristics makes Nv-CATH a promising molecule template for the development of novel antimicrobial and antibiotic-resistant agents.

A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx.

The roles of bactericidal cathelicidins against bacterial infection have been extensively studied. However, the antibacterial property and mechanism of action of non-bactericidal cathelicidins are rarely known. Herein, a novel naturally occurring cathelicidin (PopuCATH) from tree frog (Polypedates puerensis) did not't show any direct anti-bacterial activity in vitro. Intriguingly, intraperitoneal injection of PopuCATH before bacterial inoculation significantly reduced the bacterial load in tree frogs and mice, and reduced the inflammatory response induced by bacterial inoculation in mice. PopuCATH pretreatment also increased the survival rates of septic mice induced by a lethal dose of bacterial inoculation or cecal ligation and puncture (CLP). Intraperitoneal injection of PopuCATH significantly drove the leukocyte influx in both frogs and mice. In mice, PopuCATH rapidly drove neutrophil, monocyte/macrophage influx in mouse abdominal cavity and peripheral blood with a negligible impact on T and B lymphocytes, and neutrophils, monocytes/macrophages, but not T and B lymphocytes, were required for the preventive efficacy of PopuCATH. PopuCATH did not directly act as chemoattractant for phagocytes, but PopuCATH obviously drove phagocyte migration when it was cultured with macrophages. PopuCATH significantly elicited chemokine/cytokine production in macrophages through activating p38/ERK mitogen-activated protein kinases (MAPKs) and NF-κB p65. PopuCATH markedly enhanced neutrophil phagocytosis via promoting the release of neutrophil extracellular traps (NETs). Additionally, PopuCATH showed low side effects both in vitro and in vivo. Collectively, PopuCATH acts as a host-based immune defense regulator that provides prophylactic efficacy against bacterial infection without direct antimicrobial effects. Our findings reveal a non-bactericidal cathelicidin which possesses unique anti-bacterial action, and highlight the potential of PopuCATH to prevent bacterial infection.

Cathelicidin-NV from Nanorana ventripunctata effectively protects HaCaT cells, ameliorating ultraviolet B-induced skin photoaging.

Cathelicidins are diverse effector molecules in the vertebrate immune system and are related to immune regulation, inflammatory response, wound healing, and blood vessel formation. However, little is known about their free radical scavenging ability, especially in vivo. In this study, a cathelicidin molecule (cathelicidin-NV, ARGKKECKDDRCRLLMKRGSFSYV) previously identified from the spot-bellied plateau frog (Nanorana ventripunctata) (Anura, Dicroglossidae, Dicroglossinae) by us was shown to alleviate ultraviolet B (UVB)-induced skin photoaging in mice. Cathelicidin-NV effectively suppressed cytotoxicity, DNA fragmentation, apoptosis and reduced the protein expression levels of JNK, c-Jun, and MMP-1, which are involved in the regulation of collagen degradation in HaCaT cells induced by UVB irradiation. Furthermore, cathelicidin-NV also scavenged UVB-induced intracellular reactive oxygen species (ROS). Taken together, cathelicidin-NV directly scavenged excessive intracellular ROS to protect HaCaT cells, and subsequently alleviated UVB-induced skin photoaging. This study extends reports on the antioxidant function of the cathelicidin family. In addition, the properties of cathelicidin-NV make it an excellent candidate for the prevention and treatment of UV-induced skin photoaging.

Two antimicrobial and nematicidal peptides derived from sequences encoded Picea sitchensis.

Two antimicrobial peptides (piceain 1 and 2) derived from sequences encoded Picea sitchensis are identified. Their amino acid sequences are KSLRPRCWIKIKFRCKSLKF and RPRCWIKIKFRCKSLKF, respectively. One intra-molecular disulfide bridge is formed by these two half-cysteines in both piceain 1 and 2. Antimicrobial activities of synthesized piceains against several kinds of microorganisms were tested. They showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and fungus Candida albicans but little antimicrobial activity against Bacillus subtilis. The results of nematicidal test showed they exerted strong nematicidal activities against Caenorhabditis elegans, following exposure for 5 h at concentrations as low as 10 µg/ml. They had weak hemolytic abilities against human and rabbit red cells. At the concentration of 250 µg/ml, they induced red cell hemolysis of less than 5%. Circular dichroism spectra of the two antimicrobial peptides were investigated in several solutions. Their main secondary structure components are ß-sheet and random. The current work provides a novel family of antimicrobial and nematicidal peptides with unique disulfided loop containing nine amino acid residues.

Discovery of Antioxidant Peptides from Amphibians: A Review.

In recent years, bioactive peptide drugs have attracted growing attention due to the increasing difficulty in developing new drugs with novel chemical structures. In addition, many diseases are linked to excessive oxidation in the human body. Therefore, the role of peptides with antioxidant activity in counteracting diseases related to oxidative stress is worth exploring. Amphibians are a major repository for bioactive peptides that protect the skin from biotic and abiotic stresses, such as microbial infection and radiation injury. We characterized the first amphibian- derived gene-encoded antioxidant peptides in 2008. Since then, a variety of antioxidant peptides have been detected in different amphibian species. In this work, the physicochemical properties of antioxidant peptides identified from amphibians are reviewed for the first time, particularly acquisition methods, amino acid characteristics, antioxidant mechanisms, and application prospects. This review should provide a reference for advancing the identification, structural analysis, and potential therapeutic value of natural antioxidant peptides.

A Frog Peptide Ameliorates Skin Photoaging Through Scavenging Reactive Oxygen Species.

Although many bioactive peptides have been identified from the frog skins, their protective effects and the molecular mechanisms against skin photodamage are still poorly understood. In this study, a novel 20-residue peptide (antioxidin-NV, GWANTLKNVAGGLCKMTGAA) was characterized from the skin of plateau frog Nanorana ventripunctata. Antioxidin-NV obviously decreased skin erythema, thickness and wrinkle formation induced by Ultraviolet (UV) B exposure in hairless mice. In UVB-irradiated keratinocytes (HaCaT cells) and hairless mice, it effectively inhibited DNA damage through reducing p-Histone H2A.X (γH2AX) expression, alleviated cell apoptosis by decreasing the expression of apoptosis-specific protein (cleaved caspase 3), and reduced interleukin-6 (IL-6) production via blocking UVB-activated Toll-like receptor 4 (TLR4)/p38/JNK/NF-κB signaling. In UVB-irradiated human skin fibroblasts (HSF cells) and hairless mice, it effectively restored HSF cells survival rate, and rescued α-SMA accumulation and collagen (especially type I collagen) production by restoring transforming growth factor-ß1 (TGF-ß1)/Smad2 signaling. We found that antioxidin-NV directly and rapidly scavenged intracellular and mitochondrial ROS in HaCaT cells upon UVB irradiation, and quickly eliminated the artificial free radicals, 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+). Taken together, antioxidin-NV directly and rapidly scavenged excessive ROS upon UVB irradiation, subsequently alleviated UVB-induced DNA damage, cell apoptosis, and inflammatory response, thus protecting against UVB-induced skin photoaging. These properties makes antioxidin-NV an excellent candidate for the development of novel anti-photoaging agent.

A Frog-Derived Immunomodulatory Peptide Promotes Cutaneous Wound Healing by Regulating Cellular Response.

Wound healing-promoting peptides exhibit excellent therapeutic potential in regenerative medicine. However, amphibian-derived wound healing-promoting peptides and their mechanism of action remain to be further elucidated. We hereby characterized a wound healing-promoting peptide, Ot-WHP, derived from Chinese concave-eared frog Odorrana tormota. It efficiently promoted wound healing in a mouse model of full-thickness wounds. Ot-WHP significantly increased the number of neutrophils in wounds, and modestly promoted neutrophil phagocytosis and phorbol myristate acetate (PMA)-induced neutrophil extracellular trap formation. Ot-WHP also significantly increased the number of macrophages in wound sites, and directly induced chemokine, cytokine and growth factor production in macrophages by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways. Of note, Ot-WHP did not act as a chemoattractant for neutrophils and macrophages, suggesting its chemotactic activity depends on inducing chemoattractant production in macrophages. Besides, Ot-WHP directly promoted keratinocyte migration by enhancing integrin expression and cell adhesion. In addition, Ot-WHP significantly enhanced the cross-talk between macrophages and keratinocytes/fibroblasts by promoting keratinocyte/fibroblast proliferation, and fibroblast-to-myofibroblast transition despite having no direct effects on keratinocyte/fibroblast proliferation, and fibroblast differentiation. Collectively, Ot-WHP directly elicited the production of regulatory factors in macrophages, consequently initiated and accelerated the inflammatory phase by recruiting neutrophils and macrophages to wounds, and in turn enhanced the cross-talk between macrophages and keratinocytes/fibroblasts, additionally promoted keratinocyte migration, and finally promoted cutaneous wound healing. Our findings provide a promising immunomodulator for acute wound management and new clues for understanding the mechanism of action of amphibian-derived wound healing-promoting peptides.

Anti-inflammatory activities of Aedes aegypti cecropins and their protection against murine endotoxin shock.

BACKGROUND: Mosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown. RESULTS: By searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1ß and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1ß and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS. CONCLUSIONS: The current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Purification and characterization of a novel defensin from the salivary glands of the black fly, Simulium bannaense.

BACKGROUND: Black flies (Diptera: Simuliidae) are haematophagous insects that can cause allergic reactions and act as vectors of pathogens. Although their saliva has been thought to contain a diverse array of physiologically active molecules, little information is available on antimicrobial factors in black fly salivary glands, especially no defensins have been reported so far. METHODS: A novel cationic defensin designated SibaDef 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 SibaDef was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-positive bacteria Staphylococcus aureus or Bacillus subtilis treated with SibaDef were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the expression of SibaDef mRNA in whole bodies of insects after oral infection with the bacteria S. aureus or B. subtilis. RESULTS: Surprisingly, the phylogenetic analysis of defensin-related amino acid sequences demonstrated that SibaDef is most closely related to defensins from the human body louse Pediculus humanus corporis (Anoplura: Pediculidae), rather than to other dipteran defensins. SibaDef showed potent antimicrobial activities against Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.83 µM to 2.29 µM. SEM analysis indicated that SibaDef killed microorganisms through the disruption of cell membrane integrity. The transcript levels of SibaDef in the bacteria-immunized flies increased with the time course, reaching maximum at 36 h and then slowly decreased from that time point. CONCLUSIONS: Our results indicate that SibaDef is involved in the innate humoral response of the black fly S. bannaense, and it might play a significant role in the defence against microorganisms in both sugar and blood meals.

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.