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Membrane Interactions and Biological Activity of Antimicrobial Peptides from Australian Scorpion.
Abstract
UyCT peptides are antimicrobial peptides isolated from the venom of the Australian scorpion. The activity of the UyCT peptides against Gram positive and Gram negative bacteria and red blood cells was determined. The membrane interactions of these peptides were evaluated by dye release (DR) of the fluorophore calcein from liposomes and isothermal titration calorimetry (ITC); and their secondary structure was determined by CD. Three different lipid systems were used to mimic red blood cells, E. coli and S. aureus membranes. UyCT peptides exhibited broad spectrum antimicrobial activity with low MIC for S. aureus and multi-drug resistant Gram negative strains. Peptide combinations showed some synergy enhancing their potency but not haemolytic activity. The UyCT peptides adopted a helical structure in lipid environments and DR results confirmed that the mechanism of action is by disrupting the membrane. ITC data indicated that UyCT peptides preferred prokaryotic rather than eukaryotic membranes. The overall results suggest that UyCT peptides could be pharmaceutical leads for the treatment of Gram negative multiresistant bacterial infections, especially against A. baumanni, and candidates for peptidomimetics to enhance their potency and minimize haemolysis.
... AMPs can be categorized into different families according to their primary structure, secondary structures, and functions [38]. They can be arranged in three major groups: (i) linear cysteine-free peptides with an α-helical conformation (insect cecropins, magainins, etc.), (ii) cyclic and open-ended cyclic peptides with one to four disulfide bridges (defensins, protegrin, etc.), and (iii) peptides with an over-representation of some amino acids (prolinerich, glycine-rich, histidine-rich, etc.) [39]. The first and second groups include many AMPs derived from scorpion venom. ...
... An analysis of the venom of the Australian scorpion Urodacus yaschenkoi led to the isolation of four NDBPs: UyCT1, UyCT2, UyCT3, and UyCT5. In their study, Luna-Ramírez and co-workers investigated the membrane interactions and biological activity of these peptides [39]. All the peptides were tested for their antimicrobial activity against multidrugresistant pathogens including P. aeruginosa, A. baumannii, S. aureus, and E. coli [39]. ...
... In their study, Luna-Ramírez and co-workers investigated the membrane interactions and biological activity of these peptides [39]. All the peptides were tested for their antimicrobial activity against multidrugresistant pathogens including P. aeruginosa, A. baumannii, S. aureus, and E. coli [39]. This was performed by introducing the peptide in liquid cultures of the tested strains, which included both G + and G − bacteria. ...
Scorpion venom peptides are generally classified into two main groups: the disulfide bridged peptides (DBPs), which usually target membrane-associated ion channels, and the non-disulfide bridged peptides (NDBPs), a smaller group with multifunctional properties. In the past decade, these peptides have gained interest because most of them display functions that include antimicrobial, anticancer, haemolytic, and anti-inflammatory activities. Our current study focuses on the short (9-19 amino acids) antimicrobial linear scorpion peptides. Most of these peptides display a net positive charge of 1 or 2, an isoelectric point at pH 9-10, a broad range of hydrophobicity, and a Grand Average of Hydropathy (GRAVY) Value ranging between −0.05 and 1.7. These features allow these peptides to be attracted toward the negatively charged phospholipid head groups of the lipid membranes of target cells, a force driven by electrostatic interactions. This review outlines the antimicrobial potential of short-chained linear scorpion venom peptides. Additionally, short linear scorpion peptides are in general more attractive for large-scale synthesis from a manufacturing point of view. The structural and functional diversity of these peptides represents a good starting point for the development of new peptide-based therapeutics.
... We selected AMPs from two Australian scorpion species (U. yaschenkoi and U. manicatus) because the evolution of scorpions has involved the development of venom glands producing peptides and proteins that can efficiently kill insect prey [24,26]. Certain scorpion AMPs are also active against human pathogenic bacteria [21,28]. We used three insect AMPs (cecropin A, apidaecin and stomoxyn), as well as a synthetic insecticide (imidacloprid) and antibiotic (rifampicin) as controls to evaluate the scorpion AMPs. ...
... The observed insecticidal and antibacterial activities of scorpion AMPs against A. pisum and its bacterial symbionts are supported by earlier research in which indolicidin, an AMP from bovine neutrophils, showed activity against the green peach aphid Myzus persicae (Sulzer) and also affected the bacteriome [50]. Furthermore, scorpion AMPs (UyCT3, UyCT5, Uy192, Um4, D11) and indolicin showed activity against Escherichia coli, which is closely related to B. aphidicola, providing further support for our observations [20,21,28,51,52]. ...
... All peptides were amidated at the C-terminus. The scorpion AMPs selected for this study were chosen based on their activity against human pathogenic bacteria [21,28], and those with low MICs and haemolytic values were preferred (Table 3). Three insect AMPs were tested as controls: cecropin A from the moth Hyalophora cecropia (Linnaeus), apidaecin from the bumblebee Bombus pascuorum (Scopoli), and stomoxyn from the stable fly Stomoxys calcitrans (Linnaeus) [37,39,40]. ...
Aphids are severe agricultural pests that damage crops by feeding on phloem sap and vectoring plant pathogens. Chemical insecticides provide an important aphid control strategy, but alternative and sustainable control measures are required to avoid rapidly emerging resistance, environmental contamination, and the risk to humans and beneficial organisms. Aphids are dependent on bacterial symbionts, which enable them to survive on phloem sap lacking essential nutrients, as well as conferring environmental stress tolerance and resistance to parasites. The evolution of aphids has been accompanied by the loss of many immunity-related genes, such as those encoding antibacterial peptides, which are prevalent in other insects, probably because any harm to the bacterial symbionts would inevitably affect the aphids themselves. This suggests that antimicrobial peptides (AMPs) could replace or at least complement conventional insecticides for aphid control. We fed the pea aphids (Acyrthosiphon pisum) with AMPs from the venom glands of scorpions. The AMPs reduced aphid survival, delayed their reproduction, displayed in vitro activity against aphid bacterial symbionts, and reduced the number of symbionts in vivo. Remarkably, we found that some of the scorpion AMPs compromised the aphid bacteriome, a specialized organ that harbours bacterial symbionts. Our data suggest that scorpion AMPs holds the potential to be developed as bio-insecticides, and are promising candidates for the engineering of aphid-resistant crops.
... Here, we describe the functional characterization of natural and engineered versions of the scorpion AMPs UyCT1, UyCT3, UyCT5, Uy17, Uy192 and Uy234 from Urodacus yaschenkoi, and Um2, Um3, Um4 and Um5 from U. manicatus [17][18][19][20]. The AMPs were tested for antifungal, antibacterial, hemolytic and cytotoxic activities in order to identify more potent and selective versions with a higher therapeutic index. ...
... This was more diverse, containing sequences from U. yaschenkoi (UyCT3, UyCT5, Uy17, Uy192), U. manicatus (Um2, Um5, Um4), Pandinus cavimanus (Pantinin-2, Pantinin-3), Heterometrus petersii (Hp1090), Opisthacanthus madagascariensis (IsCT1, IsCT2), Mesobuthus eupeus (Meucin-18) and Vaejovis mexicanus (VmCT1, VmCT2). Third clade comprised the longer AMPs (17)(18)(19) residues, whereas all the other AMPs were 13-14 residues in length). All the AMPs in this clade are secreted from scorpions representing the Buthidae family, except ctriporin which represents the Chaerilidae family and Uy234 from the Urodacidae family. ...
... One class of compounds, known as "non-disulfide-bridged peptides", comprises short peptides with diverse properties including antimicrobial activity, lytic activity and the potentiation of bradykinin and cell signaling [24]. These peptides are considered to belong to the broader category of antimicrobial peptides (AMPs), an important component of innate immunity in many different organisms, due to their ability to inhibit the growth of pathogens including multidrug-resistant bacteria at MIC values as low as 1 µM [18,19,25]. Scorpion venom AMPs therefore provide a rich source of potential new antimicrobial compounds that could be used to address the increasing prevalence of pathogens resistant to conventional antibiotics. ...
The spread of multidrug-resistant human pathogens has drawn attention towards antimicrobial peptides (AMPs), which are major players in the innate immune systems of many organisms, including vertebrates, invertebrates, plants and microbes. Scorpion venom is an abundant source of novel and potent AMPs. Here, we investigated natural and engineered AMPs from the scorpions Urodacus yaschenkoi and U. manicatus to determine their antimicrobial spectra as well as their hemolytic/cytotoxic activity. None of the AMPs were active against fungi, but many of them were active at low concentrations (0.25-30 µM) against seven different bacteria. Hemolytic and cytotoxic activities were determined using pig erythrocytes and baby hamster kidney cells, respectively. The amino acid substitutions in the engineered AMPs did not inhibit cytotoxicity, but reduced hemolysis and therefore increased the therapeutic indices. The phylogenetic analysis of scorpion AMPs revealed they are closely related and the GXK motif is highly conserved. The engineered scorpion AMPs offer a promising alternative for the treatment of multidrug-resistant bacterial infections and could be modified further to reduce their hemolytic/cytotoxic activity.
... At lower peptide doses, there was a preference for phosphatidic acid (PA) over phosphatidylcholine (PC); although, this preference was not observed with increasing peptide concentrations. Almaaytah et al. also identified two antibacterial peptides (AamAP1 and AamAP2) from the venom of Androctonus amoreuxi [103] . Both peptides include 18 aa and vary by only two residues, with leucine replaced by proline at posi-tion 2 and phenylalanine replaced by isoleucine at position 17. ...
... Although none of the peptides was highly effective (MICs 20−150 μmol·L −1 ), AamAP1 was more effective against Gram-negative bacteria than Gram-positive ones. In the same dosage range, both were similarly induced significant hemolysis of red blood cells [103] . Notably, by focusing on the venom of Urodacus yaschenkoi an in-depth study using transcriptome techniques and a combination of MS/MS was employed, aiming to identify a variety of AMPs [104,105] . ...
Evolution and natural selection have endowed animal venoms, including scorpion venoms, with a wide range of pharmacological properties. Consequently, scorpions, their venoms, and/or their body parts have been used since time immemorial in traditional medicines, especially in Africa and Asia. With respect to their pharmacological potential, bioactive peptides from scorpion venoms have become an important source of scientific research. With the rapid increase in the characterization of various components from scorpion venoms, a large number of peptides are identified with an aim of combating a myriad of emerging global health problems. Moreover, some scorpion venom-derived peptides have been established as potential scaffolds helpful for drug development. In this review, we summarize the promising scorpion venoms-derived peptides as drug candidates. Accordingly, we highlight the data and knowledge needed for continuous characterization and development of additional natural peptides from scorpion venoms, as potential drugs that can treat related diseases.
... In this approach, the replacement of serine and glycine residues by the cationic amino acid lysine in strategic positions lead to better antimicrobial activity of StigA8 and StigA18 in relation to the prototype molecule. The increase in cationicity, helical conformation, and hydrophobic moment has been reported to be important characteristics for a better action on different microorganisms such as bacteria, viruses, and protozoa [64,65,68]. However, the molecular dynamics have shown that the peptides (native and analogs), when they come into contact with the bacterial phospholipid bilayer, generally acquire a more extensive random conformation. ...
... StigA8 and StigA18 revealed conformational flexibility, stability in extreme pH and temperature by the circular dichroism technique, being able to return to its initial structure after heating and cooling. Similar characteristics were observed in other T. stigmurus scorpion peptides, such as Stigmurin and its analogs StigA6, StigA16, StigA25, and StigA31 [29,71], as well as TsAP-2 [32], and in other scorpion species such as Meucin 13 and Meucin-18 from the scorpion Mesobuthus eupeus [72]; Pandinin-1 and Pandinin-2 of the scorpion Pandinus Imperator [73]; and UyCT1, UyCT2, UyCT3, and UyCT5 of the Urodacus yaschenkoi [68]. Therefore, StigA8 and StigA18 have become convenient molecules in the development of antimicrobial drugs, which can be used in different infection sites (stomach, intestinal, dermatological, among others) [74]. ...
Multidrug-resistant bacterial infections are a threat to public health worldwide, which boosts the urgent need for pharmacological research for new drugs. Although the peptides without disulfide bridges from scorpions have shown antimicrobial action, usually their toxicity hamper their pharmacological application. Stigmurin is a non-hemolytic cationic peptide from Tityus stigmurus venom with antibacterial effect and toxicity on normal cells. In this approach, the conformational changes and stability of two Stigmurin analog peptides, named StigA8 and StigA18, were evaluated by circular dichroism, as well as the mechanism of interaction with bacterial membranes in silico. In addition, the in vitro and in vivo antibacterial activity and the action against the biofilm formed by multidrug-resistant Staphylococcus aureus were investigated. StigA8 (+4) and StigA18 (+5) revealed the ability to change their structural conformation depending on the medium composition, and high stability at different temperatures and pH conditions. Both analog peptides showed greater ability to interact with bacterial membranes in silico when compared to the native one. StigA8 and StigA18 demonstrated low hemolytic action, with non-toxic effect on G. mellonella larvae up to 120 mg/kg. StigA8 and StigA18 presented a broad spectrum of antibacterial action in vitro, especially against multidrug-resistant clinical isolates. The analog peptides (7.5 µM) also reduced the biofilm biomass of multidrug-resistant S. aureus, as well as increased the larval survival of the Galleria mellonella infected larvae. Therefore, StigA8 and StigA18 showed a beneficial potential in the treatment of bacterial infections, constituting promising bioactive components for the development of new antimicrobial agents.
... Aunque el mecanismo de acción de la mayoría de estos PAMs se desconoce, se ha propuesto que actúan como formadores de poros causando rotura de la membrana celular [88]. La característica anfipática de los PAMs hace que algunos péptidos se inserten en la membrana formando poros, o bien, formando un "tapete" donde se agrupan en zonas cargadas positivas de la membrana [76,[89][90][91]. Además de la muerte celular por permeabilidad de la membrana, se han descrito otros mecanismos de acción que intervienen en la síntesis de proteínas, síntesis de pared celular, daño al ADN y activación de autolisinas. ...
... Acinetobacter baumannii [89], y además se ha demostrado que estos péptidos requieren amidación de su residuo C-terminal para ser activos. En este trabajo, se evaluó la estructura, la actividad hemolítica y bactericida de cuatro PAMs sintéticos de U. ...
The rise of MDR bacteria represents a significant public health problem worldwide. Currently, the available antimicrobial therapies are not sufficiently effective in eliminating MDR pathogens, so it is essential to study antimicrobials with new mechanisms of action that favor the control of these pathogens.
The use of antimicrobial peptides (AMPs) has been suggested as an alternative in the treatment of infectious diseases because the mechanisms of action differ from those of broad-spectrum antibiotics; unfortunately, only a few antimicrobial peptides have been approved for therapeutic use. Antimicrobial peptides from scorpion venoms are an example of bioactive substances with potential for use against MDR bacteria.
In this study, we determined the minimum bactericidal concentration (MBC) of three AMPs from Urodacus yaschenkoi scorpion (Uy234, Uy17, and Uy192) against the clinical isolates of MDR bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs. All peptides showed high bactericidal activity, with the highest activity against beta-hemolytic Streptococcus. The hemolytic activity of these peptides against human erythrocytes was significantly lower than that of previously reported scorpion AMPs, and the α-helical structure was confirmed by circular dichroism (CD); suggesting that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A precise index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria.
On the other hand, an antagonistic bacterium that produces inhibitory substances with interesting properties was evaluated. This bacterium, molecularly identified in this work as Pseudomonas protegens EMM-1, is capable of antagonizing a broad spectrum of MDR bacteria and phytopathogenic fungi. Besides, it has been possible to partially characterize bacteriocin-like inhibitory substances which are highly effective in inhibiting the growth of beta-hemolytic Streptococcus.
n this work, it was proven that P. protegens EMM-1 carries genes involved in the production of some interesting metabolites such as bacteriocin LlpA, 2,4-diacetylphloroglucinol, and pyoluteorin. The production of inhibitory substances in liquid medium was confirmed, and it is inferred that the production of these substances is influenced by the presence of oxygen, a probable reason because it did not produce inhibitory activity in the liquid medium before. These characteristics give P. protegens EMM-1 the possibility of being used both clinically and agronomically, as it is an ideal candidate for the control of MDR bacteria and phytopathogenic fungi.
... Furthermore, we demonstrated that all structural restraints were accurate in spite of peptide dynamics. Here, we solved the structures of P1 and P3 in a binary 4:1 POPC/Chol mixture, as a way to mimic the zwitterionic nature and cholesterol content found in the outer leaflet of mammalian cell membranes (43). ...
... As a result, the insertion and tilting of the peptide needed to elicit membrane disruption can occur at lower P/L than in homogeneous bilayers. Similar behaviors were observed for melittin (71), as well as for pardaxin (72) and scorpion HDPs (43) in binary POPC/Chol mixtures. Ld domains in phase separated (raft-containing) mixtures were shown to be targets for a diverse set of other antimicrobial peptides (73,74), and also to harbor both fiber and pore formation by islet amyloid polypeptide (IAPP) (75). ...
The host-defense peptide (HDP) piscidin 1 (P1), isolated from the mast cells of striped bass, has potent activities against bacteria, viruses, fungi, and cancer cells and also can modulate the activity of membrane receptors. Given its broad pharmacological potential, here we used several approaches to better understand its interactions with multicomponent bilayers representing models of bacterial (phosphatidyl-ethanolamine/phosphatidylglycerol [PE/PG]) and mammalian (phosphatidyl-choline/cholesterol [PC/Chol]) membranes. Using solid-state NMR, we solved the structure of P1 bound to PC/Chol and compared it with that of P3, a less potent homolog. The comparison disclosed that although both peptides are interfacially bound and α-helical, they differ in bilayer orientations and depths of insertion, and these differences depended on bilayer composition. Although Chol is thought to make mammalian membranes less susceptible to HDP-mediated destabilization, we found that Chol does not affect the permeabilization effects of P1. X-ray diffraction experiments revealed that both piscidins produce a demixing effect in PC/Chol membranes by increasing the fraction of the Chol-depleted phase. Furthermore, P1 increased the temperature required for the lamellar-to-hexagonal phase transition in PE bilayers, suggesting that it imposes a positive membrane curvature. Patch-clamp measurements on the inner Escherichia coli membrane showed that P1 and P3 at concentrations sufficient for their antimicrobial activity substantially decrease the activating tension for bacterial mechanosensitive channels. This indicated that piscidins can cause lipid redistribution and restructuring in the microenvironment near proteins. We conclude that the mechanism of piscidin's antimicrobial activity extends beyond simple membrane destabilization, helping to rationalize its broader spectrum of pharmacological effects.
... As shown in Figure 2, aurein and citropin display a rather straight helical secondary structure, while maculatin and caerin are bent, due to the proline residues and highly curved DPC micelle environment used in the NMR studies. 9−12 CD spectroscopy reports the helical content of aurein and UyCT5, two 13 residue peptides with similar charge, to be 74% and 55%, 13,15 respectively, in the presence of neutral vesicles, with similar values of 70% and 65%, respectively, in the presence of anionic vesicles. The hemolytic concentration, however, for aurein is over 68 μM 8 and that for UyCT5 14 μM. ...
... The hemolytic concentration, however, for aurein is over 68 μM 8 and that for UyCT5 14 μM. 15 The toxin UyCT5 also exhibits significantly greater antimicrobial activity than the AMP aurein, which indicates that peptide length, charge, and hydrophobicity criteria are insufficient to explain the difference in membrane-disrupting activity between the two peptides. At three amino acids longer, the AMP citropin, which caries a + 2 charge, has slightly greater helical content in anionic sodium dodecyl sulfate micelles (78%) than UyCT5 and aurein. ...
The structure-function relationship for a family of antimicrobial peptides (AMPs) from the skin of Australian tree frogs is discussed and compared with that of peptide toxins from bee and Australian scorpion venoms. Although these membrane-active peptides induce a similar cellular fate by disrupting the lipid bilayer integrity, their lytic activity is achieved via different modes of action, which are investigated in relation to amino acid sequence, secondary structure, and membrane lipid composition. In order to better understand what structural features govern the interaction between peptides and lipid membranes, cell-penetrating peptides (CPPs), which translocate through the membrane without compromising its integrity, are also discussed. AMPs possess membrane lytic activities that are naturally designed to target the cellular membrane of pathogens or competitors. They are extremely diverse in amino acid composition and often show specificity against a particular strain of microbe. Since our antibiotic arsenal is declining precariously in the face of the rise in multiantibiotic resistance, AMPs increasingly are seen as a promising alternative. In an effort to understand their molecular mechanism, biophysical studies of a myriad of AMPs have been reported, yet no unifying mechanism has emerged, rendering difficult the rational design of drug leads. Similarly, a wide variety of cytotoxic peptides are found in venoms, the best known being melittin, yet again, predicting their activity based on a particular amino acid composition or secondary structure remains elusive. A common feature of these membrane-active peptides is their preference for the lipid environment. Indeed, they are mainly unstructured in solution and, in the presence of lipid membranes, quickly adsorb onto the surface, change their secondary structure, eventually insert into the hydrophobic core of the membrane bilayer, and finally disrupt the bilayer integrity. These steps define the molecular mechanism by which these membrane-active peptides lyse membranes. The last class of membrane-active peptides discussed are the CPPs, which translocate across the lipid bilayer without inducing severe disruption and have potential as drug vehicles. CPPs are typically highly charged and can show antimicrobial activity by targeting an intracellular target rather than via a direct membrane lytic mechanism. A critical aspect in the structure-function relationship of membrane-active peptides is their specific activity relative to the lipid membrane composition of the cell target. Cell membranes have a wide diversity of lipids, and those of eukaryotic and prokaryotic species differ greatly in composition and structure. The activity of AMPs from Australian tree frogs, toxins, and CPPs has been investigated within various lipid systems to assess whether a relationship between peptide and membrane composition could be identified. NMR spectroscopy techniques are being used to gain atomistic details of how these membrane-active peptides interact with model membranes and cells, and in particular, competitive assays demonstrate the difference between affinity and activity for a specific lipid environment. Overall, the interactions between these relatively small sized peptides and various lipid bilayers give insight into how these peptides function at the membrane interface.
... To combat this silent issue that currently affects the world [1], different approaches are being addressed that range from vaccine development to the development of new bioactive materials and the search for new active principles with antimicrobial activity that will make it difficult to develop drug resistance [2][3][4]. Molecules of natural origin such as peptides are promising [5] because, among other features, they present multiple activities against several microorganisms, action mechanisms that evade the development of antimicrobial resistance [6], and a good biosafety and biodegradability profile, which makes them an interesting option for the development of new antimicrobial drugs. ...
... They lost their elongated shape and became completely round and vacuolated, and the movement of their flagellum was disturbed (24). Hadrurin is the anti-microorganism peptide of scorpion venom after scorpine B regarded as the first anti-microbial peptide with a spiral structure (25). The analogue of scorpine B named Shiva-3 prepared by chemical synthesis can interfere with P. berghei sporogonia. ...
Background: The present study determined the effect of the fractions obtained from Hemiscorpius lepturus scorpion venom on the tachyzoite of Toxoplasma gondii.
Methods: The fractions of dried venom of He. lepturus scorpion of Khuzestan Province, southern Iran in 2019 were isolated through gel filtration chromatography, and then tachyzoites were exposed to fractions of venom at different concentrations. Trypan blue counting and MTT were applied to assay tachyzoite viability, and the inhibition of the cellular growth of fractions in Vero cells was evaluated.
Results: The maximum effect on tachyzoite was observed in fraction 5 of venom. To further separate the protein, fraction 5 was used in high-performance liquid chromatography assay to purify its proteins. Based on the results of HPLC of fraction 5, among which the second peak, a peptide with
... This has already been demonstrated by the discovery of many AMPs in the venom of, e.g., spiders, scorpions, snakes, insects, and cone snails [4][5][6][7][8][9][10]. Many of the AMPs discovered in venoms were found to be effective against a wide range of bacteria, including Methicillin-resistant Staphylococci e. g. [11,12]. For some AMPs, an apparent potential for the application against multi-resistant bacteria was already described. ...
Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseu-doscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin1 1−21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid speci-ficity has not been proven. The N-and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.
... We found that Satanin 1, 2, and Curvicin have an α-helical structure, characteristic of the cecropin family, consistent with other Scarabaeidae cecropins, like Oxysterlins 1-3 and other α-helix peptides like Magainin, Cecropin A [60] and Australian scorpion antimicrobial peptides [25,[61][62][63][64][65][66]. The potential therapeutic application of AMPs and HDPs relies on their ability to effectively kill microbial cells without exhibiting significant toxicity toward mammalian cells. ...
Antibiotic resistance is an increasing global problem and therapeutic alternatives to traditional antibiotics are needed. Antimicrobial and host defense peptides represent an attractive source for new therapeutic strategies, given their wide range of activities including antimicrobial, antitumoral and immunomodulatory. Insects produce several families of these peptides, including cecropins. Herein, we characterized the sequence, structure, and biological activity of three cecropins called satanin 1, 2, and curvicin, found in the transcriptome of two dung beetle species Dichotomius satanas and Onthophagus curvicornis. Sequence and circular dichroism analyses show that they have typical features of the cecropin family: short length (38 - 39 amino acids), positive charge, and amphipathic α-helical structure. They are active mainly against Gram-negative bacteria (3.12 - 12.5 μg/mL), with low toxicity on eukaryotic cells resulting in high therapeutic indexes (TI > 30). Peptides also showed effects on TNFα production in LPS-stimulated PBMCs. The biological activity of Satanin 1, 2 and Curvicin makes them interesting leads for antimicrobial strategies.
... Similarly to other linear antimicrobial peptides, Stigmurin does not present conformational preferences in aqueous environments as observed by CD spectroscopy [29]. In comparison with other antimicrobial peptides, Stigmurin adopts a well-defined active helical conformation when in contact with membrane-mimicking environments, and our hypothesis is that it demonstrates a profile similar to that observed in magainins, phylloseptins [52,80], and NDBPs present in scorpion venoms [81,82]. As the structure in a membrane-mimetic such as TFE: H 2 O solution is an important piece of information to understand the mechanism of action, we decided to investigate the three-dimensional structure of Stigmurin. ...
Infectious diseases and the rapid development of pathogens resistant to conventional drugs are a serious global public health problem, which motivates the search for new pharmacological agents. In this context, cationic peptides without disulfide bridges from different species of scorpion venom have been the target of scientific studies due to their multifunctional activities. Stigmurin is a linear peptide composed of 17 amino acid residues (Phe-Phe-Ser-Leu-Ile-Pro-Ser-Leu-Val-Gly-Gly-Leu-Ile-Ser-Ala-Phe-Lys-NH2), which is present in the venom gland of the scorpion Tityus stigmurus. Here we present investigations of the in vitro antioxidant action of Stigmurin together with the in vivo antibacterial and healing activity of this peptide in a wound infection model induced by Staphylococcus aureus. In addition, we have reports for the first time of the three-dimensional structure determined by NMR spectroscopy of a peptide without disulfide bridges present in scorpion venom from the Tityus genus. Stigmurin showed hydroxyl radical scavenging above 70 % at 10 μM and antibiotic action in the skin wound, reducing the number of viable microorganisms by 67.2 % on the 7 day after infection. Stigmurin (1 μg / μL) increased the retraction rate of the lesion, with wound area reduction of 43 % on the second day after skin injury, which indicates its ability to induce tissue repair. Stigmurin in trifluoroethanol:water exhibited a random conformation at the N-terminus region (Phe1 to Pro6), with a helical structure from Ser7 to Phe16. This structural information, allied with the multifunctional activity of Stigmurin, makes it an attractive candidate for the design of novel therapeutic agents.
... Urodacus yaschenkoi is an Australian scorpion that has been characterized and contains an arsenal of biomolecules with diverse functions, including AMPs. Previous studies have reported that three of these peptides (UyCT1, UyCT3 and UyCT5) have been effective in Scorpion peptides and their bactericidal activity against clinical isolates of multidrug-resistant bacteria inhibiting a broad spectrum of bacteria [27], including MDR pathogens such as Acinetobacter baumannii [42], and these peptides required the amidation of the C-terminal residue to be active. ...
Scorpion venom peptides represent a novel source of antimicrobial peptides (AMPs) with broad-spectrum activity. In this study, we determined the minimum bactericidal concentration (MBC) of three scorpion AMPs, Uy234, Uy17, and Uy192, which are found in the venomous glands of the Urodacus yaschenkoi scorpion, against the clinical isolates of multidrug-resistant (MDR) bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs with the aim of obtaining higher antimicrobial activity. All peptides tested showed high antimicrobial activity against MDR clinical isolates, with the highest activity against β-hemolytic Streptococcus strains. The hemolytic activity was determined against human red blood cells and was significantly lower than that of previously reported AMPs. The α-helical structure of the four AMPs was confirmed by circular dichroism (CD). These results suggest that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A clear index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria.
... The hemolytic activity of peptides was determined by measuring the amount of hemoglobin released by human red blood cells (hRBCs) as described in a previous study (Luna-Ramírez et al., 2014). The hRBCs were washed and resuspended in 10 mM PBS (pH 7.4) to attain a dilution of ∼2% heamatocrit. ...
With the emergence of multidrug-resistant bacteria, antimicrobial peptides (AMPs) are regarded as potential alternatives to traditional antibiotics or chemicals. We designed and synthesized six derivatives of bactenecin (L2C3V10C11, RLCRIVVIRVCR), including R2F3W10L11 (RRFRIVVIRWLR), R2W3W10R11 (RRWRIVVIRWRR), K2W3V10R11 (RKWRIVVIRVRR), W2R3V10R11 (RWRRIVVIRVRR), W2K3K10R11 (RWKRIVVIRKRR), and K2R3R10K11 (RKRRIVVIRRKR), by amino acid substitution to increase the net charge and reduce hydrophobicity gradually. The bioactivity and mechanisms of action of the designed peptides were investigated. The results indicated that the antimicrobial activity of the designed peptides was higher than that of bactenecin. The hemolytic activity and cytotoxicity of the designed peptides were significantly lower than those of bactenecin. The designed peptides exhibited a wide range of antimicrobial activity against food-pathogens, particularly peptides K2W3V10R11 and W2R3V10R11; in addition, the activity was maintained under physiological salt and heat conditions. Mechanism studies indicated that AMPs interacted with negatively charged bacterial cell membranes, resulting in the destruction of cell membrane integrity by increasing membrane permeability and changing transmembrane potential, leading to cell death. The present study suggested that peptides K2W3V10R11 and W2R3V10R11 exhibited potential as alternatives to traditional antibiotics or chemicals for the treatment of food-pathogens. These findings lead to the development of a potential method for the design of novel AMPs.
... This in combination with the anionic POPG further amplifies the void for peptide insertion in the headgroup region. Recent reports have hinted at POPE and POPG as the better choice for lipids used for making membrane mimics of certain Gram-negative bacteria [60][61][62]. The incongruity between the zwitterionic phospholipids and the anionic phospholipids affects the compatibility of their combinations. ...
Cationic antimicrobial peptides (AMPs) are emerging as effective alternatives to conventional therapeutics that are used against the ever-rising number of multidrug-resistant microbial strains. Most studies established the peptide's amphipathicity and electrostatic interaction with the membrane as the basis for their antimicrobial
effect. However, the interplay between stoichiometric ratio of lipids, local geometry, diverse physico-chemical properties of the host membranes and antimicrobial peptide efficacy is still poorly understood. In the present study, we investigate the mechanism of interaction of VG16KRKP (VARGWKRKCPLFGKGG), a novel AMP designed from dengue-virus fusion peptide, with bacterial/fungal membrane mimics. Fluorescence-dye leakage assays show that membrane disruption is not solely induced by electrostatic interaction. We show that despite electrostatic screening, the initial interaction of the peptide with the membrane can be driven by stoichiometric ratio of the lipids that dictates the net surface charge, amount of lipid defects and local geometry of the membrane. Solid-state 14N and 31P NMR experiments show that peptide interaction results in lowering of lipid order
around both the headgroups and acyl chains, suggesting deep peptide insertion. Further, an increase or decrease in membrane stability of the host membrane was observed in differential scanning calorimetry (DSC) thermograms, dictated by the overall stoichiometric ratio of the lipids and the sterol present. In general, our results help
understand the diverse fates of host membranes against an antimicrobial peptide.
... Scorpion venoms are a rich source of antimicrobial peptides [16,17]. In this study we have examined the mechanism of action of Smp24 (24 amino acids, charge +3), a prototypical amphipathic AMP that was identified by genomic analysis of Scorpio maurus palmatus [18]. ...
Determining the mechanism of action of antimicrobial peptides (AMPs) is critical if they are to be developed into the clinical setting. In recent years high resolution techniques such as atomic force microscopy (AFM) have increasingly been utilised to determine AMP mechanism of action on planar lipid bilayers and live bacteria. Here we present the biophysical characterisation of a prototypical AMP from the venom of the North African scorpion Scorpio maurus palmatus termed Smp24. Smp24 is an amphipathic helical peptide containing 24 residues with a charge of + 3 and exhibits both antimicrobial and cytotoxic activity and we aim to elucidate the mechanism of action of this peptide on both membrane systems.
... DBPs and NDBPs are disulfide-bridged peptides and non-disulfide-bridged peptides, respectively from 2 to 30 μM) than eukaryotic ones (K d ranging from 150 to 200 μM) as confirmed through using dye release assays and isothermal titration calorimetry. These findings suggested that UyCT peptides could be good templates to develop new antibiotics against Gram-negative multiresistant bacteria, especially A. baumannii (Luna-Ramírez et al. 2014). ...
Recently, the OMIC technologies of proteomics and transcriptomics have been extensively applied to deeply understand the structure and molecular diversity of animal venoms from various species including scorpions. During the last decade, proteomics analysis of 27 different scorpion species has been conducted and revealed the complexity of scorpion venoms. Mass fingerprinting data clearly showed noticeable differences in the number of venom molecules (ranging from 60 to 665) between scorpion species. Also, as a complementary approach, the transcriptomics analysis of venom glands from 20 different scorpion species resulted in about 73,000 assigned ESTs which mainly correspond to neurotoxins, antimicrobial peptides, housekeeping proteins, hypothetical proteins, and a large number of unassigned types of scorpion venom peptides or proteins. The employment of transcriptomics in scorpion venomics is adding new insights about venom diversity and biological processes of venom gland as well as facilitating the identification of various novel biologically active peptides. In this chapter, scorpion gland transcriptomics will be mainly discussed with a brief introduction about scorpion venom and its proteomics analysis.
... Interestingly, these peptides have a greater affinity toward prokaryotic model membrane systems (K d ranging from 2 to 30 mM) than eukaryotic ones (K d ranging from 150 to 200 mM) as confirmed through using dye release assays and isothermal titration calorimetry. These findings suggested that UyCT peptides could be good templates to develop new antibiotics against Gram-negative multiresistant bacteria, especially A. baumannii (Luna-Ramírez et al. 2014). ...
Recently, the OMIC technologies of proteomics and transcriptomics have been extensively applied to deeply understand the structure and molecular diversity of animal venoms from various species including scorpions. During the last decade, proteomics analysis of 27 different scorpion species has been conducted and revealed the complexity of scorpion venoms. Mass fingerprinting data clearly showed noticeable differences in the number of venom molecules (ranging from 60 to 665) between scorpion species. Also, as a complementary approach, the transcriptomics analysis of venom glands from 20 different scorpion species resulted in about 73,000 assigned ESTs which mainly correspond to neurotoxins, antimicrobial peptides, housekeeping proteins, hypothetical proteins, and a large number of unassigned types of scorpion venom peptides or proteins. The employment of transcriptomics in scorpion venomics is adding new insights about venom diversity and biological processes of venom gland as well as facilitating the identification of various novel biologically active peptides. In this chapter, scorpion gland transcriptomics will be mainly discussed with a brief introduction about scorpion venom and its proteomics analysis.
... One of the most abundant components identified from U. yaschenkoi transcriptome were the antimicrobial peptides (21%) whereas for C. noxius transcriptome only one isogroup containing 6 reads was similar to porine, an antimicrobial peptide. Once again, this finding was expected because non-Buthidae scorpions are a well known source of antimicrobial peptides [100][101][102][103] while Buthidae scorpions lack these compounds. ...
Australian scorpion venoms have been poorly studied, probably because they do not pose an evident threat to humans. In addition, the continent has other medically important venomous animals capable of causing serious health problems. Urodacus yaschenkoi belongs to the most widely distributed family of Australian scorpions (Urodacidae) and it is found all over the continent, making it a useful model system for studying venom composition and evolution. This communication reports the whole set of mRNA transcripts produced by the venom gland. U. yaschenkoi venom is as complex as its overseas counterparts. These transcripts certainly code for several components similar to known scorpion venom components, such as: alpha-KTxs, beta-KTxs, calcins, protease inhibitors, antimicrobial peptides, sodium-channel toxins, toxin-like peptides, allergens, La1-like, hyaluronidases, ribosomal proteins, proteasome components and proteins related to cellular processes. A comparison with the venom gland transcriptome of Centruroides noxius (Buthidae) showed that these two scorpions have similar components related to biological processes, although important differences occur among the venom toxins. In contrast, a comparison with sequences reported for Urodacus manicatus revealed that these two Urodacidae species possess the same subfamily of scorpion toxins. A comparison with sequences of an U. yaschenkoi cDNA library previously reported by our group showed that both techniques are reliable for the description of the venom components, but the whole transcriptome generated with Next Generation Sequencing platform provides sequences of all transcripts expressed. Several of which were identified in the proteome, but many more transcripts were identified including uncommon transcripts. The information reported here constitutes a reference for non-Buthidae scorpion venoms, providing a comprehensive view of genes that are involved in venom production. Further, this work identifies new putative bioactive compounds that could be used to seed research into new pharmacological compounds and increase our understanding of the function of different ion channels.
Los venenos de muchas especies de escorpiones son fuentes ricas en componentes biológicamente activos, como los péptidos antimicrobianos, biomoléculas que aún no han sido estudiados del veneno de Hadruroides charcasus. El objetivo de este artículo es evaluar la actividad antimicrobiana de los péptidos parcialmente purificados del veneno del escorpión Hadruroides. asus. A partir de 15,46 mg de proteína total del veneno del escorpión H. charcasus se purificaron parcialmente sus péptidos por medio de cromatografía de filtración en gel empleando sephadex G-75, consecutivo a una cromatografía de intercambio iónico en CM-Sephadex C-25. El peso molecular estimado de los péptidos se determinó mediante electroforesis PAGE-SDS-Tris-Tricina al 15% y la evaluación de la actividad antibacteriana y antifúngica se empleó el método de microdilución y Kirby-Bauer con cepas de Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853 y Candida albicans ATCC 10231. En la cromatografía de filtración en gel se obtuvieron 5 fracciones, de lo cual, la fracción IV presentó una concentración mínima inhibitoria de 3,6 mg/mL en S. aureus ATCC 29213 y en C. albicans ATCC 10231. De la cromatografía de intercambio se obtuvieron 7 fracciones, destacando la fracción OPDIV-5 con péptidos de 4 kDa; 5 kDa; 5,5 kDa y 6,4 kDa que presentó actividad antimicrobiana frente S. aureus ATCC 29213, E. coli ATCC 25922, P. aeruginosa ATCC 27853, C. albicans ATCC 10231.El veneno del escorpión H. charcasus presenta péptidos de naturaleza catiónica con actividad antibacteriana y antifúngica, según su actividad en las cepas evaluadas.
Methicillin-resistant staphylococci have become growing threats to human health, and novel antimicrobials are urgently needed. Natural antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics. Here, two novel cationic α-helical antimicrobial peptides, Lausporin-1 and Lausporin-2, were identified from the venom gland of the scorpion L. australasiae through a cDNA library screening strategy. Biochemical analyses demonstrated that Lausporin-1 and Lausporin-2 are cationic α-helical amphipathic molecules. Antimicrobial assays demonstrated that the two peptides possess antibacterial activities against several species of antibiotic-resistant staphylococci. Importantly, they are active against methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus capitis, with the minimum inhibitory concentrations ranging from 2.5 to 10 μg/ml. Moreover, both peptides can induce dose-dependent plasma membrane disruptions of the bacteria. In short, our work expands the knowledge of the scorpion L. australasiae venom-derived AMPs and sheds light on the potential of Lausporin-1 and Lausporin-2 in the development of novel drugs against methicillin-resistant staphylococci.
Background
Antimicrobial Peptides (AMPs) are an attractive alternative to traditional small molecule antibiotics as AMPs typically target the bacterial cell membrane. A Trp-rich peptide sequence derived from water buffalo (Bubalus bubalis), buCATHL4B was previously identified as a broad-spectrum antimicrobial peptide.
Objective
In this work, native Trp residues were replaced with other naturally occurring aromatic amino acids to begin to elucidate the importance of these residues on peptide activity.
Methods
Minimal Inhibitory Concentration (MIC) results demonstrated activity against seven strains of bacteria. Membrane and bilayer permeabilization assays were performed to address the role of bilayer disruption in the activity of the peptides. Lipid vesicle binding and quenching experiments were also performed to gain an understanding of how the peptides interacted with lipid bilayers.
Results
MIC results indicate the original, tryptophan-rich sequence, and the phenylalanine substituted sequences exhibit strong inhibition of bacterial growth. In permeabilization assays, peptides with phenylalanine substitutions have higher levels of membrane permeabilization than those substituted with tyrosine. In addition, one of the two-tyrosine substituted sequence, YWY, behaves most differently in the lowest antimicrobial activity, showing no permeabilization of bacterial membranes. Notably the antimicrobial activity is inherently species dependent, with varying levels of activity against different bacteria.
Conclusion
There appears to be little correlation between membrane permeabilization and activity, indicating these peptides may have additional mechanisms of action beyond membrane disruption. The results also identify two sequences, denoted FFF and YYW, which retain antibacterial activity but have markedly reduced hemolytic activity.
In recent years, lipopeptides (LPs) have attracted a lot of attention in the pharmaceutical industry due to their broad-spectrum of antimicrobial activity against a variety of pathogens and their unique mode of action. This class of compounds has enormous potential for application as an alternative to conventional antibiotics and for pest control. Understanding how LPs work from a structural and biophysical standpoint through investigating their interaction with cell membranes is crucial for the rational design of these biomolecules. Various analytical techniques have been developed for studying intramolecular interactions with high resolution. However, these tools have been barely exploited in lipopeptide-lipid interactions studies. These biophysical approaches would give precise insight on these interactions. Here, we reviewed these state-of-the-art analytical techniques. Knowledge at this level is indispensable for understanding LPs activity and particularly their potential specificity, which is relevant information for safe application. Additionally, the principle of each analytical technique is presented and the information acquired is discussed. The key challenges, such as the selection of the membrane model are also been briefly reviewed.
The venoms of spiders and scorpions contain a variety of chemical compounds. Antimicrobial peptides (AMPs) from these organisms were first discovered in the 1990s. As of May 2015, there were 42 spider's and 63 scorpion's AMPs in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP). These peptides have demonstrated broad or narrow-spectrum activities against bacteria, fungi, viruses, and parasites. In addition, they can be toxic to cancer cells, insects and erythrocytes. To provide insight into such an activity spectrum, this article discusses the discovery, classification, structure and activity relationships, bioinformatics analysis, and potential applications of spider and scorpion AMPs. Our analysis reveals that, in the case of linear peptides, spiders use both glycine-rich and helical peptide models for defense, whereas scorpions use two distinct helical peptide models with different amino acid compositions to exert the observed antimicrobial activities and hemolytic toxicity. Our structural bioinformatics study improves the knowledge in the field and can be used to design more selective peptides to combat tumors, parasites, and viruses.
Objectives:
In searching for new spermicidal microbicides for use in the prevention of unplanned pregnancy and sexually transmitted infections (STIs) we investigated the spermicidal and cytotoxicity activities of the antimicrobial peptide sarcotoxin Pd.
Methods:
Washed sperm from 10 healthy, normal volunteers was treated with different concentrations of sarcotoxin Pd. Sperm motility and morphology were assessed at 0, 0.3, 5, 10 and 15 min. The cytotoxicity of sarcotoxin Pd in normal human cervical HeLa cells was measured. Percentage cell survival was expressed as the number of live cells in the test group.
Results:
The cytotoxic effect of sarcotoxin Pd was concentration-dependent. Significant cytotoxicity was observed at concentrations above 24 μg/ml. Sarcotoxin Pd immobilised 100% of spermatozoa at a dose of 90 and 80 μg/ml after 0.3 and 5 min, respectively, and immobilised 50% of spermatozoa after 15 min at lower doses. Sarcotoxin Pd inhibited sperm motility in a dose-dependent manner. The peptide immobilised sperm within 20 s at its maximal effective concentration of 90 μg/ml.
Conclusions:
Sarcotoxin Pd appears to be a good candidate for a contraceptive agent in the prevention of unplanned pregnancy and STIs.
Inhibition of dipeptidyl peptidase-IV (DPP-IV) is used as a means to regulate post-prandial serum glucose in type 2 diabetics. The effect of drug (Sitagliptin®)/peptide and binary peptide mixtures on DPP-IV inhibition was studied using an isobole approach. Five peptides (Ile-Pro-Ile-Gln-Tyr, Trp-Lys, Trp-Pro, Trp-Arg and Trp-Leu), having DPP-IV half maximum inhibitory concentration values (IC50) < 60 μM and reported to act through different inhibition mechanisms, were investigated. The dose response relationship of Sitagliptin : peptide (1 : 0, 0 : 1, 1 : 852, 1 : 426 and 1 : 1704 on a molar basis) and binary Ile-Pro-Ile-Gln-Tyr : peptide (1 : 0, 0 : 1, 1 : 1, 1 : 2 and 2 : 1 on a molar basis) mixtures for DPP-IV inhibition was characterised. Isobolographic analysis showed, in most instances, an additive effect on DPP-IV inhibition. However, a synergistic effect was observed with two Sitagliptin : Ile-Pro-Ile-Gln-Tyr (1 : 426 and 1 : 852) mixtures and an antagonistic effect was seen with one Sitagliptin : Trp-Pro (1 : 852) mixture, and three binary peptide mixtures (Ile-Pro-Ile-Gln-Tyr : Trp-Lys (1 : 1 and 2 : 1) and Ile-Pro-Ile-Gln-Tyr : Trp-Leu (1 : 2)). The results show that Sitagliptin and food protein-derived peptides can interact, thereby enhancing overall DPP-IV inhibition. Combination of Sitagliptin with food protein-derived peptides may help in reducing drug dosage and possible associated side-effects.
The need for new antimicrobial agents is becoming one of the most urgent requirements in modern medicine. The venoms of many different species are rich sources of biologically active components and various therapeutic agents have been characterized successfully including antimicrobial peptides (AMPs). Due to their potent activity, low resistance rates and unique mode of action, AMPs have recently received much attention. This review focuses on AMPs from the venoms of scorpions and examines all classes of AMPs found to date. It gives details of their biological activities with reference to peptide structure. The review examines the mechanism of action of AMPs and with this information, suggests possible mechanisms of action of less well characterised peptides. Finally, the review examines current and future trends of scorpion AMP research, by discussing recent successes obtained through proteomic and transcriptomic approaches.
Background:
Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity.
Methods:
Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured.
Results:
A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26-Α29), and K4N Δ(K12-Q18; Ν26-Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25μM and 5 to 25μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900μM for their EC50, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells.
Conclusions:
These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections.
General significance:
Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.
Scorpion venom glands produce a large variety of bioactive peptides. This communication reports the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared with venomous glands of the Brazilian scorpion Opisthacanthus cayaporum (Ischnuridae). Two main types of components were identified: peptides with toxin-like sequences and proteins involved in cellular processes. Using the expressed sequence tag (EST) strategy 118 clones were identified, from which 61 code for unique sequences (17 contigs and 44 singlets) with an average length of 531 base-pairs (bp). These results were compared with those previously obtained by the proteomic analysis of the same venom, showing a considerable degree of similarity in terms of the molecular masses expected and DNA sequences found. About 36% of the ESTs correspond to toxin-like peptides and proteins with identifiable open reading frames (ORFs). The cDNA sequencing results also show the presence of sequences whose putative products correspond to a scorpine-like component; three short antimicrobial peptides; three K(+)-channel blockers; and an additional peptide containing 78 amino acid residues, whose sequence resembles peptide La1 from another Ischnuridae scorpion Liocheles australiasiae, thus far with unknown function.
The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php) has been updated and expanded. It now hosts 1228 entries with 65 anticancer, 76 antiviral (53 anti-HIV), 327 antifungal
and 944 antibacterial peptides. The second version of our database (APD2) allows users to search peptide families (e.g. bacteriocins,
cyclotides, or defensins), peptide sources (e.g. fish, frogs or chicken), post-translationally modified peptides (e.g. amidation,
oxidation, lipidation, glycosylation or d-amino acids), and peptide binding targets (e.g. membranes, proteins, DNA/RNA, LPS or sugars). Statistical analyses reveal
that the frequently used amino acid residues (>10%) are Ala and Gly in bacterial peptides, Cys and Gly in plant peptides,
Ala, Gly and Lys in insect peptides, and Leu, Ala, Gly and Lys in amphibian peptides. Using frequently occurring residues,
we demonstrate database-aided peptide design in different ways. Among the three peptides designed, GLK-19 showed a higher
activity against Escherichia coli than human LL-37.
The D enantiomers of three naturally occurring antibiotics--cecropin A, magainin 2 amide, and melittin--were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% alpha-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D- and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.
The α-helix of the designed amphipathic peptide antibiotic LAH4(KKALLALALHHLAHLALHLALALKKA-NH2) strongly interacts with phospholipid membranes. The peptide is oriented parallel to the membrane surface under acidic conditions,
but transmembrane at physiological pH (Bechinger, B. (1996) J. Mol. Biol. 263, 768–775). LAH4 exhibits antibiotic activities against Escherichia coli and Bacillus subtilis; the peptide does not, however, lyse human red blood cells at bacteriocidal concentrations. The antibiotic activities of
LAH4 are 2 orders of magnitude more pronounced at pH 5 when compared with pH 7.5. Although peptide association at low pH is reduced
when compared with pH 7.5, the release of the fluorophore calcein from large unilamellar 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol vesicles is more pronounced at pH values where LAH4 adopts an orientation along the membrane surface. The calcein release experiments thereby parallel the results obtained in
antibiotic assays. Despite a much higher degree of association, calcein release activity of LAH4 is significantly decreased for negatively charged membranes. Pronounced differences in the interactions of LAH4 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine membranes also become apparent when the mechanisms of dye release are investigated. The results
presented in this paper support models in which antibiotic activity is caused by detergent-like membrane destabilization,
rather than pore formation by helical peptides in transmembrane alignments.
Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin,
and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids
that encoded two β-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and
sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single β-lactamase, was 150 kb in length. The cefotaxime resistance
gene that encodes the TLA-1 β-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5α. Sequencing of thebla
TLA-1 gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to
class A β-lactamases: 70SXXK,130SDN, and 234KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A β-lactamase from Chryseobacterium(Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A β-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 ofSalmonella typhimurium; and 39% identity with CepA ofBacteroides fragilis. The partially purified TLA-1 β-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed
cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam,
tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum β-lactamase of Ambler class A.
The antimicrobial, insecticidal, and hemolytic properties of peptides isolated from the venom of the predatory ant Pachycondyla goeldii, a member of the subfamily Ponerinae, were investigated. Fifteen novel peptides, named ponericins, exhibiting antibacterial
and insecticidal properties were purified, and their amino acid sequences were characterized. According to their primary structure
similarities, they can be classified into three families: ponericin G, W, and L. Ponericins share high sequence similarities
with known peptides: ponericins G with cecropin-like peptides, ponericins W with gaegurins and melittin, and ponericins L
with dermaseptins. Ten peptides were synthesized for further analysis. Their antimicrobial activities against Gram-positive
and Gram-negative bacteria strains were analyzed together with their insecticidal activities against cricket larvae and their
hemolytic activities. Interestingly, within each of the three families, several peptides present differences in their biological
activities. The comparison of the structural features of ponericins with those of well-studied peptides suggests that the
ponericins may adopt an amphipathic α-helical structure in polar environments, such as cell membranes. In the venom, the estimated
peptide concentrations appear to be compatible with an antibacterial activity in vivo. This suggests that in the ant colony, the peptides exhibit a defensive role against microbial pathogens arising from prey
introduction and/or ingestion.
Thirty-one strains of Klebsiella pneumoniae (including 10 duplicates) from 21 septicemic pediatric patients (age, <2 months) were studied during a 4-month period (June
to October 1996) in which the fatality rate was 62% (13 of 21). These isolates identified by the API 20E system yielded the
same biotype. Pulsed-field gel electrophoresis experiments revealed the same clone in 31 strains. The isolates were multidrug-resistant
but were still susceptible to ciprofloxacin, imipenem, and cefoxitin. A 135-kb plasmid was harbored in all of the isolates.
No transconjugants were obtained that were resistant to ampicillin, cefotaxime, tetracycline, or gentamicin. Isoelectric focusing
for β-lactamases was performed on all strains, and three bands with pIs of 5.4, 7.6, and 8.2 were obtained. Of these, the
pI 8.2 β-lactamase had an extended-spectrum β-lactamase phenotype. PCR amplification of both TEM- and SHV-type genes was obtained.
The sequence analysis of the SHV PCR product indicated a mutation corresponding to the SHV-5 β-lactamase.
Two novel antimicrobial peptides have been identified and characterized from venom of the African scorpion Pandinus imperator. The peptides, designated pandinin 1 and 2, are alpha-helical polycationic peptides, with pandinin 1 belonging to the group of antibacterial peptides previously described from scorpions, frogs and insects, and pandinin 2 to the group of short magainin-type helical peptides from frogs. Both peptides demonstrated high antimicrobial activity against a range of Gram-positive bacteria (2.4-5.2 microM), but were less active against Gram-negative bacteria (2.4-38.2 microM), and only pandinin 2 affected the yeast Candida albicans. Pandinin 2 also demonstrated strong haemolytic activity (11.1-44.5 microM) against sheep erythrocytes, in contrast with pandinin 1, which was not haemolytic. CD studies and a high-resolution structure of pandinin 2 determined by NMR, showed that the two peptides are both essentially helical, but differ in their overall structure. Pandinin 2 is composed of a single alpha-helix with a predominantly hydrophobic N-terminal sequence, whereas pandinin 1 consists of two distinct alpha-helices separated by a coil region of higher flexibility. This is the first report of magainin-type polycationic antimicrobial peptides in scorpion venom. Their presence brings new insights into the mode of action of scorpion venom and also opens new avenues for the discovery of novel antibiotic molecules from arthropod venoms.
A new family of antimicrobial peptides was isolated from the venom of Cupiennius salei. The peptides were purified to homogeneity, and the sequence of cupiennin 1a was determined by Edman degradation: GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2. The amino acid sequences of cupiennin 1b, c, and d were obtained by a combination of sequence analysis and mass spectrometric
measurements of comparative tryptic peptide mapping. All peptides consist of 35 amino acid residues and are characterized
by a more hydrophobic N-terminal chain region and a C terminus composed preferentially of polar and charged residues. The
total charge of all cupiennins calculated under physiological conditions is +8, and their C terminus, formed by a glutamic
acid residue, is amidated. Conformational studies of the peptides revealed a high helix forming potential. Antimicrobial assays
on bacteria with cupiennin 1a, 1d, and synthesized cupiennins 1a* and 1d* showed minimal inhibitory concentrations for bacteria
in the submicromolar range. Their lytic effect on human red blood cells was lower by a factor of 8 to 14 than the highly hemolytic
melittin. Cupiennin 1a, 1b, 1d, 1a*, and 1d* showed pronounced insecticidal activity. The immediate biological effects and
the structural properties of the isolated cupiennins indicate a membrane-destroying mode of action on prokaryotic as well
as eukaryotic cells.
A user-friendly website for the analysis of protein secondary structures from Circular Dichroism (CD) and Synchrotron Radiation Circular Dichroism (SRCD) spectra has been created.
Availability: It is accessible via a server located at http://www.cryst.bbk.ac.uk/cdweb
Contact: Users may obtain information and apply for a UserID and password from cdweb@mail.cryst.bbk.ac.uk.
Supplementary information: This website forms part of the electronic resources and information service of the Centre for Protein and Membrane Structure and Dynamics (CPMSD) located at http://www.srs.dl.ac.uk/VUV/CD/cpmsd.html.
*To whom correspondence should be addressed (at Birbeck College)
Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.
Gene-encoded anti-microbial peptides (AMPs) are widespread in nature, as they are synthesized by microorganisms as well as by multicellular organisms from both the vegetal and the animal kingdoms. These naturally occurring AMPs form a first line of host defense against pathogens and are involved in innate immunity. Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the organism against environmental pathogens. They are classified into three major groups: (i) peptides with an alpha-helical conformation (insect cecropins, magainins, etc.), (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins, protegrin, etc.), and (iii) peptides with an over-representation of some amino acids (proline rich, histidine rich, etc.). Most AMPs display hydrophobic and cationic properties, have a molecular mass below 25-30 kDa, and adopt an amphipathic structure (alpha-helix, beta-hairpin-like beta-sheet, beta-sheet, or alpha-helix/beta-sheet mixed structures) that is believed to be essential to their anti-microbial action. Interestingly, in recent years, a series of novel AMPs have been discovered as processed forms of large proteins. Despite the extreme diversity in their primary and secondary structures, all natural AMPs have the in vitro particularity to affect a large number of microorganisms (bacteria, fungi, yeast, virus, etc.) with identical or complementary activity spectra. This review focuses on AMPs forming alpha-helices, beta-hairpin-like beta-sheets, beta-sheets, or alpha-helix/beta-sheet mixed structures from invertebrate and vertebrate origins. These molecules show some promise for therapeutic use.
Several hundred disulfide-bridged neurotoxic peptides have been characterized from scorpion venom; however, only few scorpion venom peptides without disulfide bridges have been identified and characterized. These non-disulfide-bridged peptides (NDBPs) are a novel class of molecules because of their unique antimicrobial, immunological or cellular signaling activities. This review provides an overview of their structural simplicity, precursor processing, biological activities and evolution, and sheds insight into their potential clinical and agricultural applications. Based on their pharmacological activities and peptide size similarity, we have classified these peptides into six subfamilies.
During 2003, 40 carbapenem-resistant Pseudomonas aeruginosa clinical isolates collected in a Mexican tertiary-care hospital were screened for metallo-β-lactamase production. Thirteen
isolates produced IMP-15, and 12 had a single pulsed-field gel electrophoresis pattern. The blaIMP-15 gene cassette was inserted in a plasmid-borne integron with a unique array of gene cassettes and was named In95.
Immune responses have been described for many different insect species'. However, it is generally acknowledged that insects lack lymphocytes and immunoglobulins and their immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropin moth, Hyalophora cecropia(1). The expression of this multicomponent system requires de novo synthesis of RNA and proteins(2) and its broad antibacterial activity is due to at least three independent mechanisms(3), the most well known of which is the insect lysozyme(4-7). However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. We recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacteral activity against Escherichia coli and several other Gram-negative bacterial. We believe that P9A and P9B play an important part in the humoral immune responses described previously(8) and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria In contrast to melittin, the main lytic component in bee venom(9) which lyses both bacteria and eukaryotic cells.
The spread of resistant bacteria, leading to untreatable infections, is a major public health threat but the pace of antibiotic discovery to combat these pathogens has slowed down. Most antibiotics were originally isolated by screening soil-derived actinomycetes during the golden era of antibiotic discovery in the 1940s to 1960s. However, diminishing returns from this discovery platform led to its collapse, and efforts to create a new platform based on target-focused screening of large libraries of synthetic compounds failed, in part owing to the lack of penetration of such compounds through the bacterial envelope. This article considers strategies to re-establish viable platforms for antibiotic discovery. These include investigating untapped natural product sources such as uncultured bacteria, establishing rules of compound penetration to enable the development of synthetic antibiotics, developing species-specific antibiotics and identifying prodrugs that have the potential to eradicate dormant persisters, which are often responsible for hard-to-treat infections.
Three novel cysteine-free venom peptides, which were referred to as Pantinin-1, Pantinin-2 and Pantinin-3, respectively, have been identified from the scorpion Pandinus imperator by cDNA cloning strategy. The precursor of each peptide consists of a signal peptide, a mature peptide with no disulfide bridges, and an acidic propeptide with a typical processing signal. Each of the three peptides is an α-helical, cationic and amphipathic molecule with 13 or 14 amino acid residues. Their amino acid sequences are homologous to those of some 13-mer antimicrobial peptides isolated from scorpions. Antimicrobial assay showed that all the three peptides possess relatively strong activities against Gram-positive bacteria and a fungus, but have very weak antimicrobial activities against Gram-negative bacteria. Toxicity assay showed that the three peptides exhibit very low or mild hemolytic activities against human red blood cells. It is interesting to see that Pantinin-3 is able to potently inhibit the growth of vancomycin-resistant Enterococcus (VRE) S13, a pathogen that can cause a number of human infections; this suggests that Pantinin-3 has great potential to be applied in the treatment of VRE infections. Our findings gain new insights into the structure/function relationships of the small linear cationic antimicrobial peptides from scorpions, and provide new templates for designing of antimicrobial agents targeting antibiotic-resistant pathogenic bacteria.
Antimicrobial peptides (AMPs), which are produced by several species including insects, other animals, micro-organisms and synthesis, are a critical component of the natural defense system. With the growing problem of pathogenic organisms resistant to conventional antibiotics, especially with the emergence of NDM-1, there is increased interest in the pharmacological application of AMPs. They can protect against a broad array of infectious agents, such as bacteria, fungi, parasite, virus and cancer cells. AMPs have a very good future in the application in pharmaceuticals industry and food additive. This review focuses on the AMPs from different origins in these recent years, and discusses their various functions and relative mechanisms of action. It will provide some detailed files for clinical research of pharmaceuticals industry and food additive in application.
A previous review summarized research prior to 2004 carried out on the bioactive host-defense peptides contained in the skin secretions of Australian anurans (frogs and toads). This review covers the extension of that research from 2004 to 2012, and includes membrane-active peptides (including antibacterial, anticancer, antifungal and antiviral peptides) together with the mechanisms by which these peptides interact with model membranes, peptides that may be classified as "neuropeptides" (including smooth muscle active peptides, opioids and immunomodulators) and peptides which inhibit the formation of nitric oxide from neuronal nitric oxide synthase. The review discusses the outcome of cDNA sequencing of signal-spacer-active peptides from an evolutionary viewpoint, and also lists those peptides for which activities have not been found to this time.
The isobole is well established and commonly used in the quantitative study of agonist drug combinations. This article reviews the isobole, its derivation from the concept of dose equivalence, and its usefulness in providing the predicted effect of an agonist drug combination, a topic not discussed in pharmacology textbooks. This review addresses that topic and also shows that an alternate method, called "Bliss independence," is inconsistent with the isobolar approach and also has a less clear conceptual basis. In its simplest application the isobole is the familiar linear plot in cartesian coordinates with intercepts representing the individual drug potencies. It is also shown that the isobole can be nonlinear, a fact recognized by its founder (Loewe) but neglected or rejected by virtually all other users. Whether its shape is linear or nonlinear the isobole is equally useful in detecting synergism and antagonism for drug combinations, and its theoretical basis leads to calculations of the expected effect of a drug combination. Numerous applications of isoboles in preclinical testing have shown that synergism or antagonism is not only a property of the two agonist drugs; the dose ratio is also important, a fact of potential importance to the design and testing of drug combinations in clinical trials.
Scorpion venom glands produce a large variety of bioactive peptides. This communication reports the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared with venomous glands of the Brazilian scorpion Opisthacanthus cayaporum (Ischnuridae). Two main types of components were identified: peptides with toxin-like sequences and proteins involved in cellular processes. Using the expressed sequence tag (EST) strategy 118 clones were identified, from which 61 code for unique sequences (17 contigs and 44 singlets) with an average length of 531 base-pairs (bp). These results were compared with those previously obtained by the proteomic analysis of the same venom, showing a considerable degree of similarity in terms of the molecular masses expected and DNA sequences found. About 36% of the ESTs correspond to toxin-like peptides and proteins with identifiable open reading frames (ORFs). The cDNA sequencing results also show the presence of sequences whose putative products correspond to a scorpine-like component; three short antimicrobial peptides; three K+-channel blockers; and an additional peptide containing 78 amino acid residues, whose sequence resembles peptide La1 from another Ischnuridae scorpion Liocheles australiasiae, thus far with unknown function.
A novel cytotoxic linear peptide, IsCT, was characterized from scorpion Opisthacanthus madagascariensis. It is a linear peptide with a molecular weight of 1501.9 Da composed of 13 amino acid residues without cysteines. MS/MS analysis showed that its C-terminal is amidated. The identity of IsCT is re-confirmed by comparing the chemical synthesized peptide with the natural one. IsCT demonstrated antimicrobial activity against both gram-positive and gram-negative bacteria and hemolytic activity to sheep red blood cells. Also, it can release histamine from rat peritoneal mast cells. The CD absorption suggested that IsCT had an α-helix configuration in aqueous TFE. IsCT is one of the shortest natural cytotoxic peptides described, and it will be a suitable model for studying peptide-lipid interactions.
Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide-lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.
Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and methicillin resistant Staphylococcus aureus (MRSA) are important nosocomial pathogens. This study reports the in vitro activity of tigecycline against 573 and 482 ESBL-producing Enterobacteriaceae and MRSA isolates, respectively. More than 94% of all tested isolates were susceptible to tigecycline; MIC(90) found was 0.25 to 2 mg/L for ESBL-producing Enterobacteriaceae and was 0.125 mg/L for MRSA. Tigecycline demonstrated excellent in vitro activity against a wide spectrum of nosocomial pathogens.
Most antimicrobial peptides have been shown to have antitumoral activity. Cecropin A, a linear 37-residue antimicrobial polypeptide produced by the cecropia moth, has exhibited cytotoxicity in various human cancer cell lines and inhibitory effects on tumor growth. In this study, we investigated the apoptosis induced by cecropin A in the promyelocytic cell line HL-60. Treatment of cells with cecropin A was characterized by loss of viability in a dose-dependent manner, lactate dehydrogenase (LDH) leakage, and modest attenuation of lysosomal integrity measured by neutral red assay. An increase of reactive oxygen species (ROS) generation, DNA fragmentation, and phosphatidylserine externalization were quantified following cecropin A exposure at a concentration of 30 microM, whereas cecropin A-induced apoptosis was independent of caspase family members, because the activity of caspase-8 and -9 were irrelevant. Nevertheless, caspase-3 activity showed a significant increase at concentrations of 20-40 microM, but a considerable reduction at 50 microM. Flow cytometry analysis revealed a dissipation of the mitochondrial transmembrane potential (Deltapsi(m)), and the accumulation of cells at sub-G1 phase measured by FACS analysis of propidium iodide (PI) stained nuclei suggested induction of apoptosis. Morphological changes measured by Hoechst 33342 or acridine orange/ethidium bromide staining showed nuclear condensation, corroborating the apoptotic action of cecropin A. Overall, these data indicate that cecropin A is able to induce apoptosis in HL-60 cells through a signaling mechanism mediated by ROS, but independently of caspase activation.
Immune responses have been described for many different insect species 1. However, it is generally acknowledged that insects lack lymphocytes and immunoglobulins and their immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia1. The expression of this multicomponent system requires de novo synthesis of RNA and proteins2 and its broad antibacterial activity is due to at least three independent mechanisms3, the most well known of which is the insect lysozyme4-7. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. We recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacteral activity against Escherichia coli and several other Gram-negative bacteria7. We believe that P9A and P9B play an important part in the humoral immune responses described previously8 and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom9 which lyses both bacteria and eukaryotic cells.
A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.
Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.
A new method is developed in which a circular dichroism (CD) spectrum is analyzed directly as a linear combination of the CD spectra (from 190 to 240 nm) of 16 proteins whose secondary structures are known from X-ray crystallography. This avoids the dilemma encountered in previous methods of trying to define single reference CD spectra that were supposed to characterize such broad and variable classes as helix, beta sheet, beta turn, and "remainder". It also permits a more accurate and flexible analysis. The usual instability in using so many parameters is automatically controlled by a simple constrained statistical regularization procedure (similar to ridge regression). Sixteen tests were made by removing 1 spectrum at a time from the set of 16 and analyzing it in terms of the other 15. The product moment correlation coefficients between the computed fractions of helix, beta sheet, beta turn, and remainder and the fractions from the X-ray data were 0.96, 0.94, 0.31,, and 0.49, respectively. Thus, the helix and beta-sheet accuracy is very good. (The corresponding values calculated by a previous method with four reference spectra were 0.85, 0.25, --0.31, and 0.46.).
Ten analogues of the 26-residue, bee venom peptide, melittin (H3N(+)-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2), were synthesized, each with 13C enrichment of a single peptide carbonyl carbon. These peptides were incorporated into bilayers of the diether lipid, ditetradecylphosphatidylcholine, aligned between stacked glass plates. Solid-state 13C nuclear magnetic resonance spectra were obtained as a function of the angle between the bilayer planes and the magnetic field of the spectrometers, and at temperatures above and below the lipid gel-to-liquid crystalline transition temperature, Tc. For bilayers aligned with the normal along the applied magnetic field there was no shift in the carbonyl resonances of residues Ile2, Ala4, Leu9, Leu13, or Ala15, with minor changes for residues Val8 and Ile20, and small changes at Val5, Leu6 and Ile17 on immobilization of the peptide below Tc. In contrast, the spectra for bilayers aligned at right angles to the field showed greatly increased anisotropy below Tc for all analogues. From these experiments it was evident that the peptide was well-aligned in the bilayers and reoriented about the bilayer normal. The observed reduced chemical shift anisotropies and the chemical shifts were consistent with melittin adopting a helical conformation with a transbilayer orientation in the lipid membranes. With the exception of Ile17, there was no apparent difference between the behaviour of residues in the two segments that form separate helices in the water-soluble form of the peptide, suggesting that in membranes the angle between the helices is greater than the 120 degrees observed in the crystal form.
A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G-50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino-acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three-dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two alpha-helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C-terminal amidated, and all D-enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N-terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions.
Two drugs that produce overtly similar effects will sometimes produce exaggerated or diminished effects when used concurrently. A quantitative assessment is necessary to distinguish these cases from simply additive action. This distinction is based on the classic pharmacologic definition of additivity that, briefly stated, means that each constituent contributes to the effect in accord with its own potency. Accordingly, the relative potency of the agents, not necessarily constant at all effect levels, allows a calculation using dose pairs to determine the equivalent of either agent and the effect by using the equivalent in the dose-response relation of the reference compound. The calculation is aided by a popular graph (isobologram) that provides a visual assessment of the interaction but also requires independent statistical analysis. The latter can be accomplished from calculations that use the total dose in a fixed-ratio combination along with the calculated additive total dose for the same effect. Different methods may be used, and each is applicable to experiments in which a single drug is given at two different sites. When departures from additivity are found, whether in "two-drug" or "two-site" experiments, the information is useful in designing new experiments for illuminating mechanisms. Several examples, mainly from analgesic drug studies, illustrate this application. Even when a single drug (or site) is used, its introduction places it in potential contact with a myriad of chemicals already in the system, a fact that underscores the importance of this topic in other areas of biological investigation.
This chapter analyzes the statistical error in isothermal titration calorimetry (ITC). ITC is widely used to obtain thermodynamics information about binding processes in chemical and biochemical systems. The instrumental responses following each injection of titrant are analyzed to obtain the heat associated with the chemical changes from that injection, and the experiment thereby produces a titration curve of heat versus extent of reaction. The dependence of the parameter standard errors on the number of titration steps is completely tied to assumptions about the nature of the error in the titrant volume. It is observed that better precision is achieved with fewer points and a larger titration range than is customarily employed in such work. The constant error model yields results that are less precise by almost an order of magnitude and in both models the relative error in equilibrium constant is more nearly constant than the absolute error. The latter observation means that van't Hoff analysis through an unweighted fit to the logarithmic form of equation is not a bad approximation, which is reassuring, since the unweighted log fit is the usual approach taken for such analyses. The possible role of correlated error in the titrant volumes is also considered.
Cannabinoid receptor agonists elicit analgesic effects in acute and chronic pain states via spinal and supraspinal pathways. We investigated whether the combination of a cannabinoid agonist with other classes of antinociceptive drugs exerted supra‐additive (synergistic) or additive effects in acute pain models in mice.
The interactions between the cannabinoid agonist CP55,940, α 2 ‐adrenoceptor agonist dexmedetomidine and μ ‐opioid receptor agonist morphine were evaluated by isobolographic analysis of antinociception in hot plate (55°C) and tail flick assays in conscious male Swiss mice. Drug interactions were examined by administering fixed‐ratio combinations of agonists (s.c.) in 1 : 1, 3 : 1 and 1 : 3 ratios of their respective ED 50 fractions.
CP55,940, dexmedetomidine and morphine all caused dose‐dependent antinociception. In the hot plate and tail flick assays, ED 50 values (mg kg ⁻¹ ) were CP55,940 1.13 and 0.51, dexmedetomidine 0.066 and 0.023, and morphine 29.4 and 11.3, respectively. Synergistic interactions existed between CP55,940 and dexmedetomidine in the hot plate assay, and CP55,940 and morphine in both assays. Additive interactions were found for CP55,940 and dexmedetomidine in the tail flick assay, and dexmedetomidine and morphine in both assays.
Thus, an α 2 ‐adrenoceptor agonist or μ opioid receptor agonist when combined with a cannabinoid receptor agonist showed significant synergy in antinociception in the hot plate test. However, for the tail flick nociceptive response to heat, only cannabinoid and μ opioid receptor antinociceptive synergy was demonstrated. If these results translate to humans, then prudent selection of dose and receptor‐specific agonists may allow an improved therapeutic separation from unwanted side effects.
British Journal of Pharmacology (2005) 144 , 875–884. doi: 10.1038/sj.bjp.0706045
Short cationic amphiphilic peptides with antimicrobial and/or immunomodulatory activities are present in virtually every life form, as an important component of (innate) immune defenses. These host-defense peptides provide a template for two separate classes of antimicrobial drugs. Direct-acting antimicrobial host-defense peptides can be rapid-acting and potent, and possess an unusually broad spectrum of activity; consequently, they have prospects as new antibiotics, although clinical trials to date have shown efficacy only as topical agents. But for these compounds to fulfill their therapeutic promise and overcome clinical setbacks, further work is needed to understand their mechanisms of action and reduce the potential for unwanted toxicity, to make them more resistant to protease degradation and improve serum half-life, as well as to devise means of manufacturing them on a large scale in a consistent and cost-effective manner. In contrast, the role of cationic host-defense peptides in modulating the innate immune response and boosting infection-resolving immunity while dampening potentially harmful pro-inflammatory (septic) responses gives these peptides the potential to become an entirely new therapeutic approach against bacterial infections.
The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix-hinge-helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.
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