Insect immunity. Isolation from the lepidopteran Heliothis virescens of a novel insect defensin with potent antifungal activity

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila. Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.     

A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata).

The injection of low doses of bacteria into the aquatic larvae of dragonflies (Aeschna cyanea, Odonata, Paleoptera) induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated a 38-residue peptide from this hemolymph which is strongly active against Gram-positive bacteria and also shows activity against one of the Gram-negative bacteria which was tested. The peptide is a novel member of the insect defensin family of inducible antibacterial peptides, which had so far only been reported from the higher insect orders believed to have evolved 100 million years after the Paleoptera. Aeschna defensin is more potent than defensin from the dipteran Phormia, from which its structure differs in several interesting aspects, which are discussed in the paper.     

Acaloleptins A: inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa

We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem.     

Isolation, characterization and chemical synthesis of a new insect defensin from Chironomus plumosus (Diptera)

Injection of low doses of bacteria into the aquatic larvae of the dipteran insect Chironomus plumosus induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated two 36-residue peptides from this hemolymph which are active against Gram-positive bacteria. The peptides are novel members of the insect defensin family and their sequences present marked differences with those of insect defensins isolated from other dipteran species. We have developed a method for efficient renaturation of this cysteine-rich molecule and obtained a highly pure synthetic Chironomus defensin.     

Isolation, gene expression and solution structure of a novel moricin analogue, antibacterial peptide from a lepidopteran insect, Spodoptera litura

An antibacterial peptide was isolated from a lepidopteran insect, Spodoptera litura. The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ionization-time of flight mass (MALDI-TOF MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopetran insects. Thus, the peptide was designated S. litura (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue-specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-simulated annealing calculation. The tertiary structure revealed a long alpha-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in B. mori but also in other lepidopteran insects forming a gene family.     

Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-arginine-rich antibacterial peptides.

We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin-P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.     

The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide.

Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.     

Role of amino acid residues within the disulfide loop of thanatin,a potent antibiotic peptide

Thanatin, a 21-residue peptide, is an inducible insect peptide with a broad range of activity against bacteria and fungi. It has a C-terminal disulfide loop, like the frog skin secretion antimicrobial peptides of the brevinin family. In this study, we tried to find the effect of a number of amino acids between the disulfide bond. Thanatin showed stronger antibacterial activity to Gram negative bacteria than other mutants, except Th1; whereas, the mutant peptides with deletion had higher activity to Gram positive bacteria than thanatin. An increase in the number of amino acid(s) using the alanine residue decreased the antibacterial activity in all of the bacteria. Th1 with deletion of threonine at position 15 (Thr(1)(2)) showed similar antibacterial activity against Gram-negative bacteria, but had higher activity against the Gram positive bacteria. In order to study the structure-function relationship, we measured liposome disruption by the peptides and CD spectra of the peptides. Th1 also showed the highest liposome leaking activity and alpha-helical propensity in the sodium dodecyl sulfate solution, compared with other peptides. Liposome disruption activity was closely correlated with the anti-Gram positive bacterial activity. All of the peptides showed no hemolytic activity. Th1 was considered to be useful as an antimicrobial peptide with broad spectrum without toxicity     

Viresin. A novel antibacterial protein from immune hemolymph of Heliothis virescens pupae.

Immune hemolymph was collected from fifth instar larvae and 1-day-old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N-terminal amino acid sequence of 12-kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12-kDa protein was negative for immunoblotting with anti-synthetic cecropin B antibody. We have named the novel 12-kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram-negative bacteria including E. cloacae but not against Gram-positive bacteria.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.     

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins

A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.     

Antibacterial activity of multiple antigen peptides homologous to a loop region in human lactoferrin.

An 11-residue peptide (FQWQRNMRKVR) homologous to just over half the loop region of human lactoferricin is thought to be responsible for antimicrobial properties of human lactoferricin. Multiple antigen peptides (MAP) of the 11-residue peptide exerted significant antibacterial effects against a broad spectrum of bacteria including MRSA. More than eight branching was favourable for increasing its antibacterial activity. Our report shows a novel possibility for MAP to increase the activity of antibiotic peptides other than simply to stimulate antibody production, as reported so far.     

Mapping of Apidaecin Regions Relevant for Antimicrobial Activity and Bacterial Internalization

Apidaecins are 18–20-residue long proline-rich peptides expressed in insects as part of the innate immune system. They are very active against Gram-negative bacteria, especially Enterobacteriaceae. The C-terminal sequence PRPPHPRL is highly conserved, whereas the N-terminal region is variable. By replacing all 18 residues of apidaecin 1a and apidaecin 1b individually by alanine (Ala-scan), we have shown that single mutations in the C-terminal half of the peptides drastically reduced and mostly abolished the antibacterial activity against Escherichia coli. Conversely, substitutions in the N-terminal eight residues produced no, or only minor effects. The activity loss was correlated to the ability of apidaecin 1b and its mutants to enter Gram-negative bacteria, most likely because they no longer bind to a protein transporter. This assumed binding, however, was not inhibited by truncated apidaecin peptides added at tenfold higher concentrations. Interestingly, the antibacterial activity of full length apidaecin 1b was enhanced about four times by addition of a N-terminally truncated apidaecin peptide [11–18]-apidaecin 1b, as indicated by lower MIC-values against E. coli, although the short 5(6)-carboxyfluorescein-labeled peptide did not enter the bacteria. In contrast, the activity against the Gram-positive bacterium Micrococcus luteus was not located in the C-terminal sequence of apidaecins 1a and b, but depended mostly on the presence of all four basic residues.     

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.