The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: identification of a potent anti-Candida peptide.

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

Alanine scanning analysis and structure-function relationships of the frog-skin antimicrobial peptide temporin-1Ta.

The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti-infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog-skin temporins, because of their short size (10-14 residues long) and their unique features. In particular, temporin-1Ta has the following properties: (i) it is mainly active on Gram-positive bacteria; (ii) it can synergize, when combined with temporin-1Tl, in inhibiting both gram-negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptide's antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin-based lead structures for the production of new anti-infective agents.

Structure-activity relationship, conformational and biological studies of temporin L analogues.

Temporins are naturally occurring peptides with promising features, which could lead to the development of new drugs. Temporin-1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human erythrocytes and this fact makes the design of synthetic analogues with a higher therapeutic index vital.We studied the structure-activity relationships of a library of TL derivatives focusing on the correlation between the α-helix content of the peptides, the nature of their cationic residues, and their antibacterial/antiyeast/hemolytic activities. We found that the percentage of helicity of TL analogues is directly correlated to their hemolytic activity but not to their antimicrobial activity. In addition, we found that the nature of positively charged residues can affect the biological properties of TL without changing the peptide's helicity. It is noteworthy that a single amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-association. Last, we identified a novel analogue with properties that make it an attractive topic for future research.

Membrane interaction and antibacterial properties of two mildly cationic peptide diastereomers, bombinins H2 and H4, isolated from Bombina skin.

Bombinins H are mildly cationic antimicrobial peptides isolated from the skin of the anuran genus Bombina, the fire-bellied toad. Some members of this peptide family coexist in skin secretions as diastereomers in which a single D: -amino acid (alloisoleucine or leucine) is incorporated as a result of the post-translational modification of the respective gene-encoded L-amino acid. Here we report on the antimicrobial properties and membrane interactions of bombinins H2 and H4. The latter differs from H2 by the presence of a D-alloisoleucine at the second N-terminal position. Specifically, we have evaluated the antimicrobial activity of H2 and H4 against a large panel of reference and clinical isolates of Gram-negative and Gram-positive bacteria; performed membrane permeation assays on both intact cells and model membranes (lipid monolayers and liposomes) mimicking the composition of the plasma membrane of Gram-negative/positive bacteria; used biochemical tools, such as trypsin-encapsulated liposomes and capillary electrophoresis, to monitor the peptides' ability to translocate through the membrane of liposomes mimicking Escherichia coli inner membrane. The results revealed interesting relationships between the presence of a single D: -amino acid in the sequence of an antimicrobial peptide and its target microbial cell selectivity/membrane-perturbing activity.

Evidence that prokineticin receptor 2 exists as a dimer in vivo.

Prokineticins are proteins that regulate diverse biological processes including gastrointestinal motility, angiogenesis, circadian rhythm, and innate immune response. Prokineticins bind two closed related G-protein coupled receptors (GPCRs), PKR1 and PKR2. In general, these receptors act as molecular switches to relay activation to heterotrimeric G-proteins and a growing body of evidence points to the fact that GPCRs exist as homo- or heterodimers. We show here by Western-blot analysis that PKR2 has a dimeric structure in neutrophils. By heterologous expression of PKR2 in Saccharomyces cerevisiae, we examined the mechanisms of intermolecular interaction of PKR2 dimerization. The potential involvement of three types of mechanisms was investigated: coiled-coil, disulfide bridges, and hydrophobic interactions between transmembrane domains. Characterization of differently deleted or site-directed PKR2 mutants suggests that dimerization proceeds through interactions between transmembrane domains. We demonstrate that co-expressing binding-deficient and signaling-deficient forms of PKR2 can re-establish receptor functionality, possibly through a domain-swapping mechanism.

Anti-Pseudomonas activity of frog skin antimicrobial peptides in a Caenorhabditis elegans infection model: a plausible mode of action in vitro and in vivo.

The emergence of multidrug-resistant (MDR) microorganisms makes it increasingly difficult to treat infections. These infections include those associated with Pseudomonas aeruginosa, which are hard to eradicate, especially in patients with a compromised immune system. Naturally occurring membrane-active cationic antimicrobial peptides (CAMPs) serve as attractive candidates for the development of new therapeutic agents. Amphibian skin is one of the richest sources for such peptides, but only a few studies on their in vivo activities and modes of action have been reported. We investigated (i) the activity and mechanism underlying the killing of short CAMPs from frog skin (e.g., temporins and esculentin fragments) on an MDR clinical isolate of P. aeruginosa and (ii) their in vivo antibacterial activities and modes of action, using the minihost model of Caenorhabditis elegans. Our data revealed that in vivo, both temporin-1Tb and esculentin(1-18) were highly active in promoting the survival of Pseudomonas-infected nematodes, although temporin-1Tb did not show significant activity in vitro under the experimental conditions used. Importantly, esculentin(1-18) permeated the membrane of Pseudomonas cells within the infected nematode. To the best of our knowledge, this is the first report showing the ability of a CAMP to permeate the microbial membrane within a living organism. Besides shedding light on a plausible mode of action of frog skin CAMPs in vivo, our data suggest that temporins and esculentins would be attractive molecules as templates for the development of new therapeutics against life-threatening infections.

Expression of Bv8 in Pichia pastoris to identify structural features for receptor binding.

Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.

Esculentin-1b(1-18)--a membrane-active antimicrobial peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in Escherichia coli.

Antimicrobial peptides constitute one of the main classes of molecular weapons deployed by the innate immune system of all multicellular organisms to resist microbial invasion. A good proportion of all antimicrobial peptides currently known, numbering hundreds of molecules, have been isolated from frog skin. Nevertheless, very little is known about the effect(s) and the mode(s) of action of amphibian antimicrobial peptides on intact bacteria, especially when they are used at subinhibitory concentrations and under conditions closer to those encountered in vivo. Here we show that esculentin-1b(1-18) [Esc(1-18)] (GIFSKLAGKKLKNLLISG-NH(2)), a linear peptide encompassing the first 18 residues of the full-length esculentin-1b, rapidly kills Escherichia coli at the minimal inhibitory concentration. The lethal event is concomitant with the permeation of the outer and inner bacterial membranes. This is in contrast to what is found for many host defense peptides, which do not destabilize membranes at their minimal inhibitory concentrations. Importantly, proteomic analysis revealed that Esc(1-18) has a limited ability to modify the bacterium's protein expression profile, at either bactericidal or sublethal concentrations. To the best of our knowledge, this is the first report on the effects of an antimicrobial peptide from frog skin on the proteome of its bacterial target, and underscores the fact that the bacterial membrane is the major target for the killing mechanism of Esc(1-18), rather than intracellular processes.

The Bv8 gene from Bombina orientalis: molecular cloning, genomic organization and functional characterization of the promoter.

Bv8 is a secreted peptide from Bombina variegata skin glands with a molecular mass close to 8kDa that is conserved in fish, amphibians and mammals. Bv8 has diverse regulatory roles, including an involvement in hematopoiesis and immunomodulation. Here we report the genomic organization of the gene from Bombina orientalis coding for the Bv8 homolog (Bo8). It contains three exons separated by two large introns. Several putative transcription factor binding sites have been identified in the promoter sequence. Functional analysis of this region was performed using a yeast genetic system. The results indicate that the transcription factors AP-1, NF-kappaB and NFAT are involved in the regulation of the expression of Bo8. Hence, amphibians are a useful model for the study of transcriptional regulation of all Bv8 homologs.

Esculentin 1-21: a linear antimicrobial peptide from frog skin with inhibitory effect on bovine mastitis-causing bacteria.

Mastitis, or inflammation of the mammary gland, is the most common and expensive illness of dairy cows throughout the world. Although stress and physical injuries may give rise to inflammation of the udders, infections by bacteria or other microorganisms remain the major cause, and infusion of antibiotics is the main treatment approach. However, the increased emergence of multidrug-resistant pathogens and the production of milk contaminated with antibiotics has become a serious threat in the livestock. Hence, there is an urgent need for the discovery of new therapeutic agents with a new mode of action. Gene-encoded AMPs, which represent the first line of defence in all living organisms, are considered as promising candidates for the development of new anti-infective agents. This paper reports on the antibacterial activities in vitro and in an animal model, of the frog skin AMP esculentin 1-21 [Esc(1-21)], along with a plausible mode of action. Our data revealed that this peptide (i) is highly potent against the most common mastitis-causing microbes (e.g. Streptococcus agalactiae); and (ii) is active in vivo, causing a visible regression of the clinical stage of mastitis in dairy cows, after 1 week of peptide treatment. Biophysical characterisation revealed that the peptide adopts an alpha-helical structure in microbial mimicking membranes and is able to permeate the membrane of S. agalactiae in a dose-dependent manner. Overall, these data suggest Esc(1-21) as an attractive AMP for the future design of new antibiotics to cure mastitis in cattle.

In vitro bactericidal activity of the N-terminal fragment of the frog peptide esculentin-1b (Esc 1-18) in combination with conventional antibiotics against Stenotrophomonas maltophilia.

In this study the bactericidal effect of the N-terminal fragment of the frog skin peptide esculentin-1b [Esc(1-18)] in combination with clinically used antimicrobial agents was evaluated against Stenotrophomonas maltophilia, either in standard conditions (phosphate buffer) or in the presence of human serum. A synergistic bactericidal effect was observed after a 24h incubation when combinations of Esc(1-18) and amikacin or colistin were used against clinical strains of S. maltophilia with or without resistance to these antibiotics, both in buffer and in the presence of serum. An indifferent effect was observed when the peptide was combined with levofloxacin or ceftazidime. A synergistic effect was also observed at earlier time points when the peptide was used in combination with colistin. Sequential exposure of bacterial cells to Esc(1-18) and amikacin or colistin, or vice versa, indicated that while Esc(1-18) and colistin cooperated in enhancing the bactericidal effect of their combination, when Esc(1-18) was combined with amikacin, the peptide had a major role in initiating the bactericidal effect, while amikacin was required for the subsequent effector phase. Altogether, the results obtained indicate that exposure of S. maltophilia to sub-bactericidal concentrations of Esc(1-18) increases its susceptibility to amikacin or colistin and may also render resistant strains susceptible to these antibiotics.

Bombinins, antimicrobial peptides from Bombina species.

The skin secretions of Bombina species contain peptides and small proteins with interesting biological properties. These include bombesin, thyrotropin releasing hormone, BSTI and Bv8. In this review, the biosynthesis and antimicrobial activity of two groups of peptides, bombinins and bombinins H, are described. To date, these have only been found in Bombina skin. They are derived from common precursors containing one or two bombinin copies at the amino and a single bombinin H at the carboxyl end. Bombinins are active against Gram-positive and Gram-negative bacteria and fungi but virtually inactive in haemolysis assays. Conversely, bombinins H have lower bactericidal activities but lyse erythrocytes. In the skin secretions, bombinins H are present in two sizes with either 20 or 17 amino acids. Moreover, they occur as epimers with either an L- or a D-amino acid at position 2. An enzyme catalyzing this inversion of chirality of an amino acid in peptide linkage has been isolated from Bombina skin secretions. In different tests, also with different stages of the life cycle of Leishmania parasites, the d-forms were found to be more active. Biophysical studies have yielded some insight into the different behaviours of the epimers in model membranes.

Lipopolysaccharide, a key molecule involved in the synergism between temporins in inhibiting bacterial growth and in endotoxin neutralization.

Lipopolysaccharide (LPS) is the major structural component of the outer membrane of Gram-negative bacteria and shields them from a variety of host defense factors, including antimicrobial peptides (AMPs). LPS is also recognized by immune cells as a pathogen-associated molecular pattern and stimulates them to secrete pro-inflammatory cytokines that, in extreme cases, lead to a harmful host response known as septic shock. Previous studies have revealed that a few isoforms of the AMP temporin, produced within the same frog specimen, can synergize to overcome bacterial resistance imposed by the physical barrier of LPS. Here we found that temporins can synergize in neutralizing the LPS-induced macrophage activation. Furthermore, the synergism between temporins, to overcome the protective function of LPS as well as its endotoxic effect, depends on the length of the polysaccharide chain of LPS. Importantly, mode of action studies, using spectroscopic and thermodynamic methods, have pointed out different mechanisms underlying the synergism of temporins in antimicrobial and anti-endotoxin activities. To the best of our knowledge, such a dual synergism between isoforms of AMPs from the same species has not been observed before, and it might explain the ability of such amphibians to resist a large repertoire of microorganisms.

Comparative analysis of the bactericidal activities of amphibian peptide analogues against multidrug-resistant nosocomial bacterial strains.

Due to the widespread resistance of bacteria to the available drugs, the discovery of new classes of antibiotics is urgently needed, and naturally occurring antimicrobial peptides (AMPs) are considered promising candidates for future therapeutic use. Amphibian skin is one of the richest sources of such AMPs. In the present study we compared the in vitro bactericidal activities of five AMPs from three different species of anurans against multidrug-resistant clinical isolates belonging to species often involved in nosocomial infections (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii). The peptides tested were temporins A, B, and G from Rana temporaria; the fragment from positions 1 to 18 of esculentin 1b [Esc(1-18)] from Rana esculenta; and bombinin H2 from Bombina variegata. When they were tested in buffer, all the peptides were bactericidal against all bacterial species tested (three strains of each species) at concentrations ranging from 0.5 to 48 microM, with only a few exceptions. The temporins were found to be more active against gram-positive bacteria, especially when they were assayed in human serum; Esc(1-18) showed fast and strong bactericidal activity, within 2 to 20 min, especially against the gram-negative species, which were killed by Esc(1-18) at concentrations ranging from 0.5 to 1 microM; bombinin H2 displayed similar bactericidal activity toward all isolates. Interestingly, while the activities of the temporins and bombinin H2 were almost completely inhibited in the presence of 20% human serum, the activity of Esc(1-18) against the gram-negative species was partially preserved in the presence of 40% serum. This property renders this peptide an attractive molecule for use in the development of new compounds for the treatment of infectious diseases.

A GATA-type transcription factor regulates expression of the high-affinity iron uptake system in the methylotrophic yeast Pichia pastoris.

The ferroxidase Fet3 and the permease Ftr1 constitute a well-conserved high-affinity iron uptake system in yeast. We have investigated the mechanism of transcriptional regulation of Fet3 in the methylotrophic yeast Pichia pastoris. Isolation and functional analysis of the Fet3 promoter indicate that a GATA sequence element plays a role in iron-dependent expression of Fet3. A GATA-type transcription factor, which we have named Fep1, has been partially cloned and it is shown to belong to the family of iron-responsive fungal GATA-factors. These factors share the presence of two Cys(2)-Cys(2) zinc-finger motifs and a set of four conserved cysteines, and are involved in the regulation of siderophore biosynthesis and/or high-affinity iron uptake. Disruption of the FEP1 gene in P. pastoris leads to constitutively high expression of Fet3, irrespective of iron levels, indicating that Fep1 is a transcriptional repressor. EMSA analyses evidence that Fep1 binds to DNA only in the presence of iron.

A synergism between temporins toward Gram-negative bacteria overcomes resistance imposed by the lipopolysaccharide protective layer.

Temporins are short and homologous antimicrobial peptides (AMPs) isolated from the frog skin of Rana genus. To date, very little is known about the biological significance of the presence of closely related AMPs in single living organisms. Here we addressed this question using temporins A, B, and L isolated from Rana temporaria. We found that temporins A and B are only weakly active toward Gram-negative bacteria. However, a marked synergism occurs when each is mixed with temporin L. To shed light on the underlying mechanisms involved in these activities, we used various experimental strategies to investigate: (i) the effect of the peptides' interaction on both the viability and membrane permeability of intact bacteria and spheroplasts; (ii) their interaction with lipopolysaccharides (LPS) and the effect of LPS on the oligomeric state of temporins, alone or combining one with another; (iii) their structure in solution and when bound to LPS, by using circular dichroism and ATR-FTIR spectroscopies. Our data reveal that temporin L synergizes with A and B by preventing their oligomerization in LPS. This should promote their translocation across the outer membrane into the cytoplasmic membrane. To the best of our knowledge, this is the first study that explains how a combination of native AMPs from the same species can overcome bacterial resistance imposed by the LPS leaflet.

Interaction of antimicrobial peptide temporin L with lipopolysaccharide in vitro and in experimental rat models of septic shock caused by gram-negative bacteria.

Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used beta-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and beta-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-alpha levels, resulting in the highest survival rates.

Effect of natural L- to D-amino acid conversion on the organization, membrane binding, and biological function of the antimicrobial peptides bombinins H.

Antimicrobial peptides (AMPs) are evolutionarily old components of innate immunity found in all living pluricellular organisms. Interestingly, some organisms express families of AMPs with only a slight variation among their members, possibly to increase their spectrum of activity. Despite the growing body of knowledge about their biological activity and mode of action on bacteria, only a few of them have been tested on Leishmania, a worldwide spread protozoan pathogen, and the parameters contributing to this activity are yet to be determined. We report on the anti-Leishmania activity and mode of action of bombinins H2 and H4 isolated from the skin secretion of the frog Bombina variegata. H4, the most active, is the first natural AMP of animal origin with a single L- to D-amino acid isomerization. Membrane depolarization and membrane permeation assays, as well as electron microscopy, suggest that the lethal mechanism involves plasma membrane permeation and/or disruption. To better understand the enhanced activity of H4, we determined the peptide's structure in membranes mimicking those of mammals, bacteria, and Leishmania by using ATR-FTIR and CD spectroscopies and assessed their membrane binding by using surface plasmon resonance. The data reveal that (i) H2 but not H4 partially aggregates in membranes mimicking those of Leishmania, (ii) H2 is slightly more helical than H4 in all membranes, and (iii) H4 binds the Leishmania model membrane approximately 5-fold better than H2. This study highlights the importance of a single alpha-amino acid epimerization as a tool used by nature to modulate the activity of AMPs. In addition, our findings suggest bombinins H as potential templates for the development of new drugs with a new mode of action against Leishmania.

Biological activities of Bv8 analogues.

1 The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N-terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C-terminal domain. Two closely related G protein-coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK-R1 and PK-R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. 2 In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des-Ala-Bv8) or two (des-Ala-Val-Bv8) residues from the N-terminus. 3 des-Ala-Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca(2+)](i) transients and in causing p42/p44 MAPK phosphorylation in CHO-cells expressing PK-R1 and PK-R2. Moreover, dA-Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. 4 The deletion of the first two amino acids of Bv8 abolished any biological activity both 'in vitro' and 'in vivo'; however, des-AlaVal-Bv8 is able to antagonize the Bv8-induced hyperalgesia, binding the PK-Rs on peripheral and central projections of the primary sensitive neurons.

The yeast multicopper oxidase Fet3p and the iron permease Ftr1p physically interact.

High affinity iron uptake in yeast is carried out by a multicomponent system formed by the ferroxidase Fet3p and the iron permease Ftr1p. The currently accepted model predicts that Fet3p and Ftr1p are functionally associated, however, a structural interaction between these two proteins has not been proven yet. The methylotrophic yeast Pichia pastoris has been used to perform cross-linking studies aimed to demonstrate the existence of a Fet3p-Ftr1p complex. Cross-linking of membrane suspensions with the membrane-impermeable reagents DTSSP and BS(3) has evidenced the presence of a high molecular weight band with Fet3p oxidase activity. This band has been purified and subjected to N-terminal sequence analysis. Two sequences were found in the cross-linked species, one of which could be assigned to Fet3p and the other to Ftr1p. This is the first experimental demonstration that Fet3p and Ftr1p are physically associated.