Abstract:- Antimicrobial peptides are the short protein sequence showing activities against various kinds of microbes, including bacteria, fungus, parasites and even viruses. Most of these antimicrobial peptides are cationic, which were originated from insects. Their number distribution and diversity varies considerably and is found to achieve their importance in developing a new mode of noncytotoxic defense against these pathogens.
Introduction: -the defense system of insects against the function of various pathogen and parasites are classified in two i.e; the first line of defense is the physical which consists of the cuticle through which the insect's external body surface is made of, which protects the insect. After the physical defense has been compromised, the insect goes for the interaction between, innate humoral and the cellular immune reaction which is a complex interaction induced in the tissue and hemocoel, this results in the rapid elimination of microorganism. The component of this insect immune response is the antimicrobial peptides which are released into the hemolymph and synthesized by the fat body of insects which are equivalent to the mammalian liver.
Historical background: – antimicrobial peptides are the smaller peptides which are an evolutionarily conserved molecule with their existence persisting from, the prokaryotes to humans. During the systemic response of the pathogen, they are synthesized inside the fat body of insects. The first antimicrobial peptide was cecropin, which was discovered from Hyalophora cecropia in the 1980s by Boman’s research group. From then till now, more than 200 antimicrobial peptides have been identified in insects. In humans, the discovery of the antimicrobial proteins lysozyme was identified in the year 1922 from the nasal mucus, by the Alexander Flemming but the discovery of penicillin in the year 1928 by him, have overshadowed his own observation. But, in the 1960s, the multi-drug resistant microbial pathogens, have awakened the field of interest for AMPs as the host defense molecules. In the field of AMPs for insects, the Hoffmann’s group in the mid-1990s, have shown that the fruit fly is more susceptible to massive fungal infection when the gene of AMP synthesis, have been ablated. This has become the first evidence of the role of AMPs in insect host defense. At present, the database for Antimicrobial peptides (http://aps.unmc.edu/AP/main.php) includes more than 2500 AMPs.
Evolution and distribution of AMPs: – the evolution of the genes involved in the antimicrobial defense may have arisen by the gene duplication and then, they are diversified by the horizontal gene transfer, or by denovo creation from the noncoding sequence. From the completed genome project, it has been found that the emergence of the major AMP genes, belongs to the insect taxa. The five megadiverse insect order i.e., Coleopter, Diptera, Hemiptera, Hymenoptera and Lepidoptera that consists the major part of insect species are found to have the most different types of AMP genes. Among the diversified family of antimicrobial peptides, there are several order specific AMPs for insect species have been discovered, for example, moricin, glycine-rich gloverin, proline-rich lebocins and the antifungal cysteine-rich peptides heliomicin and gallerimycin have only been found in the Lepidoptera. Metchnikowin which is a proline-rich AMP, only found in the genus Drosophila. The coleoptericins which is a glycine- and proline-rich peptide are only found in the Coleoptera, and it was first discovered in larvae of the tenebrionid beetle Zophobas atratus, followed by similar peptides in other beetles such as Tribolium castaneum, H. axyridis and Acalolepta luxuriosa. The proline-rich peptide abaecin and the glycine rich peptide hymenoptaecin are thought to be specific for Hymenoptera and thus are found in bees, ants, wasps belonging to the genus Nasonia and another pteromalid wasp etc.
Structure and classification of AMPs: – Antimicrobial peptides are low molecular weight (below 5 kDa) proteins, which bears a net positive charge at physiological Ph. Structurally they are amphiphilic having alpha-helices or hairpin-like beta sheets or mixed structures. The AMPs in insects have a property to adopt certain structures or can contain the unique sequences, on the basis of which they can be classified into 4 groups: –
1. the α-helical peptides e.g., cecropin and moricin,
2. cysteine-rich peptides e.g., insect defensin and drosomycin,
3. proline-rich peptides e.g., apidaecin, drosocin, and
4. lebocin, and glycine-rich proteins e.g., attacin and gloverin.
Among the majority of the AMPs present, except moricin and gloverin which have been identified only in Lepidoptera, all others are located in more than one order of insect species. In the case of D. melanogaster, by using the toll and immune deficiency signalling pathway, it was found that it consist of seven classes of AMPs which includes, cecropin, attacin, defensin, drosomycin, diptericin, drosocin, and metchnikowin, among which metchnikowin is specific for D. melanogaster.
Modes of action of AMPs: – most of the insect AMPs are found to have a net positive charge with the 50 percent residues of hydrophobic amino acids. This property of the AMPs, helps them to bind with the negatively charged and lipophilic membranes of bacterial cells. This results in reflecting the abundance of acidic phospholipids in the outer leaflet as compared to the membranes of the eukaryotic cells. The binding of the AMPs results in the nonenzymatic disruption of the cell membrane via promotion of the hydrophobic residues at the outer leaflet of the membrane thus causing the expansion then thinning and finally the lysis of the membrane. Although it was found that most of the AMPs function by increasing the membrane porosity, but there are few exceptions to it. The proline-rich AMPs such as abaecin found in bumblebee are thought to interact with the intracellular targets of the bacterial chaperone network like DnaK or they can go for the protein synthesis apparatus like attacins that inhibit the synthesis of proteins belonging to the outer bacterial membrane. Apart from these, there are few AMPs which have been shown to bear the property to inhibit the cell wall synthesis by causing disturbances with the corresponding enzymes or lipid phosphatidylethanolamines, or they can cause the delocalization of bacterial cell surface proteins. Also there are few other insect AMPs which have the property to neutralize specifically virulence-associated microbial metalloproteases e.g., insect metalloprotease inhibitor (IMPI).
Properties and function of different types of AMPs found in insects: – among the various types of AMPs found in insects, the properties and functions of few of the major AMPs are discussed over here.
• CECROPINS:- it was the first insect AMP which was discovered from the larvae of the giant silk moth Hyalophora cecropia. This structure of cecropins consists of a linear α-helical peptides with tryptophan residue at the N- terminus and more hydrophobic α-helix at the C-terminus which acts against Gram-negative bacteria like Escherichia coli. Apart from these, there are few cecropins like peptides, for e.g., sarcotoxins, hyphancin, and enbocin which can act against both gram-negative as well as gram-positive bacteria. Most cecropins show amidation at the c-terminus which helps them to interact with liposomes and contribute to the broad antimicrobial activity.
• DEFENSINS: – Defensins are the cationic or basic AMPs having the small structure (~4 kDa) with six conserved cysteine residues which helps them in forming the three intramolecular disulfide bridges. These three pairs of the disulfide bridges help them to stabilize their predominant β-sheet globular structure, having an N-terminal loop and an α-helix. On the basis of their structure, defensins can be classified into three families: “classical” defensins, beta-defensins, and insect defensins. The first defensin peptide was discovered from the flesh fly Sarcophaga peregrina and Phormia terranovae which were found to be active against Gram-positive bacteria and they are distributed among the insect orders which includes ancient apterygote insects hemimetabolous orders such as Hemiptera and Odonata, and holometabolous orders such as Coleoptera, Diptera, Hymenoptera, and Lepidoptera. Although most insect defensins were having activity against gram-positive bacteria, in spite there are few which also showed their activity against gram-negative bacteria and fungi.
• ATTACINS: – attacins are the antimicrobial peptides having the molecular mass of 20-23 kDa and a varied isoelectric point(pI) of 5.7-8.3, which were purified for the first time from the hemolymph of bacteria-immunized H. cecropia pupae. On the basis of their isoelectric point, attacins were classified into two groups: the basic attacins (A–D) and acidic attacins (E and F) which although were having similar amino acid sequences, but variation in the acidic attacin from the basic attacins were observed in the contents of Asp residues which were found to be high in acidic attacin that are encoded by two separate genes. The synthesis of attacins occurs as pre-pro-proteins having a signal peptide, a pro-peptide (P domain), and an N-terminal attacin domain. These domains are being followed by the two glycine-rich domains (G1 and G2 domains). Apart from these, they consist of conserved RXXR motif, which can be recognized by the furin-like enzymes. Their presence has been marked in most of the lepidopteran species and few of the dipterans with their activity found to be against E. coli and some selected Gram-negative bacteria.
• GLOVERINS: – These are the glycine-rich basic or highly basic (pI ~8.3 or pI >9.0), heat stable, antibacterial proteins, which have been purified for the first time from the hemolymph of Hyalophora gloveri pupae. These are being basic in nature have a property to bind with lipopolysaccharide(LPS) and thus suppress the growth of E. coli through inhibiting the synthesis of outer membrane proteins and thus increasing the membrane permeability. Till now the presence of gloverins has been marked only under Lepidoptera. Like attacins, they are also synthesised as the pre-pro-protein form, with a conserved RXXR motif at the N-terminal pro-regions which is being digested by the furin-like enzyme. Their activity has been observed mainly against the E.coli having rough mutants of lipopolysaccharide(LPS), but found inactive against the strain of E.coli with smooth mutants of LPS. The activity of gloverin against E.coli, Gram positive bacteria, fungi or even a virus is under investigation regarding different lepidopteran species. Although there is a suggestion that there might be an interaction of the basic gloverin with the LPS having the negatively charged lipid A and thus showing a charge-charge interaction, due to which LPS can inhibit the activity of Gloverins, as in case of the recombinant M. sextagloverin (pI ~9.3) which does not bind to lipid A, has the ability to bind with the O-specific antigen and outer core carbohydrate moieties of LPS, Gram-positive bacterial lipoteichoic acid (LTA) and peptidoglycan (PG), and laminarin (beta-1,3-glucan)
• MORICIN: – the first isolation of moricin was done from the E. coli-immunized B. mori larvae. These are 42 residues peptide which is highly basic in nature and have been reported only in lepidopteran insects so far. These are the secretory proteins which are synthesized after the cleavage of their signal peptides, and their activity is been reported against the gram negative and gram positive bacteria with exception of G. mellonellamoricins which also show high activity against filamentous fungi and yeast. Their tertiary structure marks the presence of long α-helix having eight turns except at the N-terminal and C-terminal region where N-terminal have the amphipathic region and the C-terminal marks the hydrophobic region, which is essential for its antimicrobial activity. Their structure shows similarity with cecropin, except at the hinge region, which is absent in moricin.
• LEBOCIN: – these are the proline-rich peptide that was first isolated from the hemolymph of E. coli-immunized silkworm, B. mori. These are the O-glycosylated 32-residue peptides, sharing 41% amino acid sequence identity with the honey bee’s proline-rich peptide abaecin, which have no O-glycosylated residue. The lebocin usually originates from the precursor proteins, which are generated by their proteolytic cleavage. These precursors contain several conserved RXXR motifs which can be recognized by the furin-like enzymes. The role of Lebocins was found to be active against the Gram-negative and Gram-positive bacteria and few fungi.
Application: – The antimicrobial peptides, present in insects, have broad range of activity with the additional advantage of being noncytotoxic. These AMPs may have potential applications in case of agriculture, disease vector control, and medicine
• Use of recombinant AMPs to control vector-borne diseases :- Insect AMPs exhibits activity against some parasites which includes Plasmodium, filarial nematode, and Trypanosome. There have been two recent reviews which summarize the current progress of AMPs in antimalarial and antiparasitic peptides in which Cecropins and defensins have been shown to be active against parasites. Use of AMPs against the transgenic vectors like mosquitoes is a new approach for both, killing and blocking their parasitic transmission.
• Use of AMPs in plant biotechnology: – in plants AMPs have been engineered to confer resistance to bacterial and fungal pathogens, as in case of transgenic rice, wheat, banana, tomato, melon, peanut, tobacco and Arabidopsis where plant defensins have been expressed. Even the cecropins have been expressed in transgenic plants, which includes rice and tomato, having the capability of resistance against bacterial and fungal pathogens, similarly Metchnikowin (proline-rich peptide) has been expressed in transgenic barley.
• And finally, there are many potential therapeutic applications of AMPs in the clinical development.
Future prospects :-