A Pore Forming Toxin-like Protein Derived from Chinese Red Belly Toad Bombina maxima Triggers the Pyroptosis of Hippomal Neural Cells and Impairs the Cognitive Ability of Mice.

Toxin-like proteins and peptides of skin secretions from amphibians play important physiological and pathological roles in amphibians. ßγ-CAT is a Chinese red-belly toad-derived pore-forming toxin-like protein complex that consists of aerolysin domain, crystalline domain, and trefoil factor domain and induces various toxic effects via its membrane perforation process, including membrane binding, oligomerization, and endocytosis. Here, we observed the death of mouse hippocampal neuronal cells induced by ßγ-CAT at a concentration of 5 nM. Subsequent studies showed that the death of hippocampal neuronal cells was accompanied by the activation of Gasdermin E and caspase-1, suggesting that ßγ-CAT induces the pyroptosis of hippocampal neuronal cells. Further molecular mechanism studies revealed that the pyroptosis induced by ßγ-CAT is dependent on the oligomerization and endocytosis of ßγ-CAT. It is well known that the damage of hippocampal neuronal cells leads to the cognitive attenuation of animals. The impaired cognitive ability of mice was observed after intraperitoneal injection with 10 µg/kg ßγ-CAT in a water maze assay. Taken together, these findings reveal a previously unknown toxicological function of a vertebrate-derived pore-forming toxin-like protein in the nerve system, which triggers the pyroptosis of hippocampal neuronal cells, ultimately leading to hippocampal cognitive attenuation.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50  = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50  = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50  = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50  = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Biochemical Profile, Biological Activities, and Toxic Effects of Proteins in the Rhinella schneideri Parotoid Gland Secretion.

Parotoid glands of amphibians are known for the production of several biologically active compounds having pharmacological and toxic effects in mammals. In the present work, a protein fraction obtained from Rhinella schneideri parotoid gland (RsPP) was characterized to study its biological and toxic effects. Rhinella schneideri parotoid secretion is composed of up to 30% (w/w) of soluble proteins. Tandem mass spectrometric analysis of the RsPP identified 104 proteins, including actin, beta-actin, ribosomal proteins, catalase, galectin, and uncharacterized proteins; however, no peptidases were found, and this result was reinforced by the absence of proteolytic activity. In addition, RsPP did not exhibit pro-coagulant or antibacterial effects. However, pretreatment of mice with different doses of RsPP intraperitoneally inhibited carrageenan-induced paw edema and increased tissue myeloperoxidase activity. RsPP also reduced interleukin 1ß levels in the peritoneal cavities and cell migration in the peritoneal cavities of an animal model of carrageenan-induced peritonitis. Subchronic treatment of animals with RsPP for 7 consecutive days did not alter the serum biochemical, renal, or liver parameters. However, a significant reduction in blood leukocyte count was observed. Our results showed that R. schneideri parotoid secretion contains proteins with anti-inflammatory and slight toxic effects.

The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptide's N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

In vitro antiviral activity of dermaseptin S(4) and derivatives from amphibian skin against herpes simplex virus type 2.

Herpes simplex virus (HSV) infections have become a public health problem worldwide. The emergence of acyclovir-resistant viral strains and the failure of vaccination to prevent herpetic infections have prompted the search for new antiviral drugs. Accordingly, the present study was undertaken to synthesize chemically and evaluate Dermaseptin S(4) (S(4)), an anti-microbial peptide derived from amphibian skin, and its derivatives in terms of anti-herpetic activity. The effects of biochemical modifications on their antimicrobial potential were also investigated. The peptides were incubated together with HSV-2 on target cells under various conditions, and the antiviral effects were examined via a cell metabolic labeling method. The findings revealed that DS(4) derivatives elicited concentration-dependent antiviral activity at micromole concentrations. The biochemical modifications of S(4) allowed for the reduction of peptide cytotoxicity without altering antiviral activity. Dermaseptins were added at different times during the viral cycle to investigate the mode of antiviral action. At the highest non-cytotoxic concentrations, most of the tested derivatives were noted to exhibit high antiviral activity particularly when pre-incubated with free herpes viruses prior to infection. Among these peptides, K(4)K(20)S(4) exhibited the highest antiviral activity against HSV-2 sensitive and resistant strains. Interestingly, the antiviral activity of K(4)K(20)S(4) was effective on both acyclovir-resistant and -sensitive viruses. The findings indicate that K(4)K(20)S(4) can be considered a promising candidate for future application as a therapeutic virucidal agent for the treatment of herpes viruses.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin.

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine ß-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Analogues of the frog skin peptide alyteserin-2a with enhanced antimicrobial activities against Gram-negative bacteria.

The emergence of strains of multidrug-resistant Gram-negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin-2a (ILGKLLSTAAGLLSNL.NH2) is a cationic, α-helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin-2a while maintaining amphipathicity by the substitution Gly¹¹ → Lys enhanced the potency against both Gram-negative and Gram-positive bacteria by between fourfold and 16-fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC50=24 µM). Antimicrobial potency was increased further by the additional substitution Ser7 →Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC50=38 µM). However, the peptide containing D-lysine at positions 7 and 11 showed high potency against a range of Gram-negative bacteria, including multidrug-resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µM) but appreciably lower haemolytic activity (LC50=185 µM) and cytotoxicity against A549 human alveolar basal epithelial cells (LC50=65 µM). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics.

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, temporin-1CEb from Chinese brown frog, Rana chensinensis.

Temporin-1CEb shows antimicrobial activity against Gram-positive bacteria, but its therapeutic potential is limited by its haemolysis. In this study, eight temporin-1CEb analogues with altered cationicities and hydrophobicities were synthesized. Increasing cationicity and amphipathicity by substituting neutral and non-polar amino acid residues on the hydrophilic face of the α-helix by five or six lysines increased antimicrobial potency approximately 10-fold to 40-fold, although when the number of positive charges was increased from +6 to +7, the antimicrobial potency was not additionally enhanced. The substitution of an l-lysine with a d-lysine, meanwhile maintaining the net charge and the mean hydrophobicity values, had only a minor effect on its antimicrobial activity, whereas significantly led a decrease in its haemolytic activity. Of all the peptides, l-K6 has the best potential as an antimicrobial agent because its antimicrobial activity against both Gram-positive and Gram-negative bacteria is substantial, and its haemolytic activity is negligible. l-K6 adopts an α-helix in 50% trifluoroethanol/water and 30 mm SDS solutions. l-K6 killed 99.9% of E. coli and S. aureus at 4× MIC in 60 min, and its postantibiotic effect was >5 h. l-K6 affects the integrity of E. coli and S. aureus plasma membranes by rapidly inducing membrane depolarization.

Brevinin-2R(1) semi-selectively kills cancer cells by a distinct mechanism, which involves the lysosomal-mitochondrial death pathway.

Brevinin-2R is a novel non-hemolytic defensin that was isolated from the skin of the frog Rana ridibunda. It exhibits preferential cytotoxicity towards malignant cells, including Jurkat (T-cell leukemia), BJAB (B-cell lymphoma), HT29/219, SW742 (colon carcinomas), L929 (fibrosarcoma), MCF-7 (breast adenocarcinoma), A549 (lung carcinoma), as compared to primary cells including peripheral blood mononuclear cells (PBMC), T cells and human lung fibroblasts. Jurkat and MCF-7 cells overexpressing Bcl2, and L929 and MCF-7 over-expressing a dominant-negative mutant of a pro-apoptotic BNIP3 (DeltaTM-BNIP3) were largely resistant towards Brevinin-2R treatment. The decrease in mitochondrial membrane potential (DeltaPsim), or total cellular ATP levels, and increased reactive oxygen species (ROS) production, but not caspase activation or the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G), were early indicators of Brevinin-2R-triggered death. Brevinin-2R interacts with both early and late endosomes. Lysosomal membrane permeabilization inhibitors and inhibitors of cathepsin-B and cathepsin-L prevented Brevinin-2R-induced cell death. Autophagosomes have been detected upon Brevinin-2R treatment. Our results show that Brevinin-2R activates the lysosomalmitochondrial death pathway, and involves autophagy-like cell death.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta: Secondary structure, antimicrobial activity, and mammalian cell toxicity.

The present study reports the structural characteristics, the biological activities, and preliminary clinical investigations of three synthetic members of the dermaseptin family of antimicrobial peptides. The three peptides showed similar tendencies to form alpha-helical structures in non-polar media. The antimicrobial activity towards bacteria and fungi was determined in the micromolar concentration and the peptides did not influenced peritoneal cells viability. One of the peptides was intravenously administered in mice at concentrations similar to those of antibiotics employed in bacterial/fungal infections and it did not cause any detectable changes in cells and tissues.

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2.

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.

The antimicrobial peptide dermaseptin S4 inhibits HIV-1 infectivity in vitro.

Most of HIV-1 infections are acquired through sexual contact. In the absence of a preventive vaccine, the development of topical microbicides that can block infection at the mucosal tissues is needed. Dermaseptin S4 (DS4) is an antimicrobial peptide derived from amphibian skin, which displays a broad spectrum of activity against bacteria, yeast, filamentous fungi, and herpes simplex virus type 1. We show here that DS4 inhibits cell-free and cell-associated HIV-1 infection of P4-CCR5 indicator cells and human primary T lymphocytes. The peptide is effective against R5 and X4 primary isolates and laboratory-adapted strains of HIV-1. Its activity is directed against HIV-1 particles by disrupting the virion integrity. Increasing the number of DS4-positive charges reduced cytotoxicity without affecting the antiviral activity. The modified DS4 inhibited HIV-1 capture by dendritic cells and subsequent transmission to CD4(+) T cells, as well as HIV-1 binding on HEC-1 endometrial cells and transcytosis through a tight epithelial monolayer.

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells.

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.