Insect antimicrobial peptides and their applications

Insects are one of the major sources of antimicrobial peptides/proteins (AMPs). Since observation of antimicrobial activity in the hemolymph of pupae from the giant silk moths Samia Cynthia and Hyalophora cecropia in 1974 and purification of first insect AMP (cecropin) from H. cecropia pupae in 1980, over 150 insect AMPs have been purified or identified. Most insect AMPs are small and cationic, and they show activities against bacteria and/or fungi, as well as some parasites and viruses. Insect AMPs can be classified into four families based on their structures or unique sequences: the α-helical peptides (cecropin and moricin), cysteine-rich peptides (insect defensin and drosomycin), proline-rich peptides (apidaecin, drosocin, and lebocin), and glycine-rich peptides/proteins (attacin and gloverin). Among insect AMPs, defensins, cecropins, proline-rich peptides, and attacins are common, while gloverins and moricins have been identified only in Lepidoptera. Most active AMPs are small peptides of 20–50 residues, which are generated from larger inactive precursor proteins or pro-proteins, but gloverins (~14 kDa) and attacins (~20 kDa) are large antimicrobial proteins. In this mini-review, we will discuss current knowledge and recent progress in several classes of insect AMPs, including insect defensins, cecropins, attacins, lebocins and other proline-rich peptides, gloverins, and moricins, with a focus on structural-functional relationships and their potential applications.     

Insect immunity: isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth, Antheraea pernyi

The immune system in the Chinese oak silk moth, Antheraea pernyi, has been compared with that of the Cecropia moth which has been characterized earlier. Antibacterial activity against Escherichia coli was induced in diapausing pupae by injection of viable E. coli or Enterobacter cloacae. The activity reached a maximum on day 7-8 after which the response gradually declined. The pupae produced a set of immune proteins with P4 and P5 as major labelled components similar to that earlier found in Cecropia. The major antibacterial factor in A. pernyi was cecropin D. A procedure is described for the isolation of cecropin B and D, which is in principle similar to the one used for the isolation of the corresponding cecropins from Cecropia pupae. Amino acid sequence analyses of the A. pernyi cecropins show the D form to contain 36 amino acid residues and that both cecropins have blocked C-termini. The general structure of cecropins having a charged N-terminal region (residues 1-21) followed by a long hydrophobic stretch (residues 22-32) is well conserved. Cecropin B and D from A. pernyi differ from the corresponding proteins in Cecropia by four and three conservative amino acid replacements, respectively. The homology between the cecropins from the two insects suggests that they orginate from a single ancestral gene. The antibacterial activity was tested against nine different bacterial species. Evolutionary aspects of the cecropins are discussed.     

Peptide Sequence of an Antibiotic Cecropin from the Vector Mosquito,Aedes albopictus

We have identified a 35-amino acid antibiotic cecropin secreted by an established mosquito cell line. C7-10 cells from the vector mosquito,Aedes albopictus,were incubated with heat-killedEscherichia coli,and materials secreted into the cell culture supernatant were recovered by acid precipitation. Following batch elution from Sep-Pak C18 cartridges and further purification by reverse phase high performance liquid chromatography (RP-HPLC) a predominant peak of antibacterial activity was characterized by mass spectrometry, amino acid composition analysis, and Edman degradation, yielding the sequence GGLKKLGKKLEGVGKRVFKASEKALPVAVGIKALG. Unlike other cecropins, the peptide was not amidated at the C-terminus.Aedes albopictusCecropin A (AalCecA) is the first cecropin to be described from a mosquito vector of human disease. Consistent with the classification of mosquitoes among the Dipteran suborder Nematocera, AalCecA shares only 36% amino acid identity with cecropins fromDrosophila melanogasterand other Cyclorrhaphid flies, whose mature cecropins share 80% to 100% amino acid identity.     

Escherichia coli mutants with an altered sensitivity to cecropin D.

Cecropins are a family of small, basic antibacterial polypeptides which can be isolated from pupae of immunized Lepidoptera. They are active against both gram-negative and gram-positive bacteria. We studied a mutant of Escherichia coli, strain SB1004, which is more sensitive to cecropin D than is the parental strain. The mutant was selected as resistant to a host range mutant of a Serratia marcescens phage. When the protein composition of the outer membrane was examined, strain SB1004 and some other phage-resistant mutants were found to be deficient in the OmpC protein. It was concluded that the OmpC protein is the receptor of the phage. Strain SB1004 was found to differ from other ompC mutants in being especially sensitive to hydrophobic antibiotics and to cecropin D. Furthermore, strain SB1004 has a tendency for spontaneous autolysis. A genetic analysis showed the mutations in strain SB1004 and a suppressor mutant to map in the ompC region. The activity of cecropin D against different strains of E. coli was specifically enhanced when divalent cations were absent. No such effect was found with cecropins A and B, which are less hydrophobic than the D form.     

Cecropins as a marker of Spodoptera frugiperda immunosuppression during entomopathogenic bacterial challenge

An antimicrobial peptide (AMP) of the cecropin family was isolated by HPLC from plasma of the insect pest, Spodoptera frugiperda. Its molecular mass is 3910.9 Da as determined by mass spectrometry. Thanks to the EST database Spodobase, we were able to describe 13 cDNAs encoding six different cecropins which belong to the sub-families CecA, CecB, CecC and CecD. The purified peptide identified as CecB1 was chemically synthesized (syCecB1). It was shown to be active against Gram-positive and Gram-negative bacteria as well as fungi. Two closely related entomopathogenic bacteria, Xenorhabdus nematophila F1 and Xenorhabdus mauleonii VC01(T) showed different susceptibility to syCecB1. Indeed, X. nematophila was sensitive to syCecB1 whereas X. mauleonii had a minimal inhibitory concentration (MIC) eight times higher. Interestingly, injection of live X. nematophila into insects did not induce the expression of AMPs in hemolymph. This effect was not observed when this bacterium was heat-killed before injection. On the opposite, both live and heat-killed X. mauleonii induced the expression of AMPs in the hemolymph of S. frugiperda. The same phenomenon was observed for another immune-related protein lacking antimicrobial activity. Altogether, our data suggest that Xenorhabdus strains have developed different strategies to supplant the humoral defense mechanisms of S. frugiperda, either by increasing their resistance to AMPs or by preventing their expression during such host-pathogen interaction.     

Design, synthesis and antibacterial activity of cecropin-like model peptides

In order to investigate structure-activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C18-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.     

Antibacterial peptides designed as analogs or hybrids of cecropins and melittin.

Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.     

In vitro induction of cecropin genes--an immune response in a Drosophila blood cell line.

The Drosophila melanogaster cell line mbn-2 was explored as a model system to study insect immune responses in vitro. This cell line is of blood cell origin, derived from larval hemocytes of the mutant lethal (2) malignant blood neoplasm (1(2)mbn). The mbn-2 cells respond to microbial substances by the activation of cecropin genes, coding for bactericidal peptides. The response is stronger than that previously described for SL2 cells, and four other tested Drosophila cell lines were totally unresponsive. Bacterial lipopolysaccharide, algal laminarin (a beta-1,3-glucan), and bacterial flagellin were strong inducers, bacterial peptidoglycan fragments gave a weaker response, whereas a formyl-methionine-containing peptide had no effect. Experiments with different drugs indicate that the response may be mediated by a G protein, but not by protein kinase C or eicosanoids, and that it requires a protein factor with a high rate of turnover.     

Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli

Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.     

Clash of kingdoms or why Drosophila larvae positively respond to fungal competitors

BACKGROUND: Competition with filamentous fungi has been demonstrated to be an important cause of mortality for the vast group of insects that depend on ephemeral resources (e.g. fruit, dung, carrion). Recent data suggest that the well-known aggregation of Drosophila larvae across decaying fruit yields a competitive advantage over mould, by which the larvae achieve a higher survival probability in larger groups compared with smaller ones. Feeding and locomotor behaviour of larger larval groups is assumed to cause disruption of fungal hyphae, leading to suppression of fungal growth, which in turn improves the chances of larval survival to the adult stage. Given the relationship between larval density, mould suppression and larval survival, the present study has tested whether fungal-infected food patches elicit communal foraging behaviour on mould-infected sites by which larvae might hamper mould growth more efficiently. RESULTS: Based on laboratory experiments in which Drosophila larvae were offered the choice between fungal-infected and uninfected food patches, larvae significantly aggregated on patches containing young fungal colonies. Grouping behaviour was also visible when larvae were offered only fungal-infected or only uninfected patches; however, larval aggregation was less strong under these conditions than in a heterogeneous environment (infected and uninfected patches). CONCLUSION: Because filamentous fungi can be deadly competitors for insect larvae on ephemeral resources, social attraction of Drosophila larvae to fungal-infected sites leading to suppression of mould growth may reflect an adaptive behavioural response that increases insect larval fitness and can thus be discussed as an anti-competitor behaviour. These observations support the hypothesis that adverse environmental conditions operate in favour of social behaviour. In a search for the underlying mechanisms of communal behaviour in Drosophila, this study highlights the necessity of investigating the role of inter-kingdom competition as a potential driving force in the evolution of spatial behaviour in insects.     

The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity

Cecropin D was synthesized by solid-phase methods and shown to be homogeneous and of correct composition and molecular weight. It was indistinguishable from natural cecropin D and constitutes a structure proof for this peptide. Several analogs of cecropin D were synthesized and used to draw conclusions about the structural features contributing to antibacterial activity. They included [Lys1]cecropin D, [Gln3, Leu4] cecropin D, and cecropin D-(9-37). It was concluded that a strongly basic NH2-terminal segment is a prerequisite for antibacterial activity. A hybrid analog cecropin A-(1-11) D-(12-37) was designed and predicted to have enhanced potency. It was found to be 5 to 55 times as active as cecropin D against six of the bacteria tested and was slightly more active than cecropin A. However, against Bacillus subtilis Bs11 the analog was 6 times more active than cecropin A.     

The immune response in Drosophila: pattern of cecropin expression and biological activity.

Cecropins are antibacterial peptides, induced in Drosophila as part of the humoral immune response to a bacterial invasion. We have used the cloned Drosophila cecropin genes CecA1, A2 and B as probes to study the developmental and tissue specific regulation of this response. The genes are strongly expressed in fat body and hemocytes after injection of bacteria, the CecA genes being much more active than CecB in the fat body. All parts of the fat body and 5-10% of the hemocytes are involved in this response. CecA1 and A2 are most active in larvae and adults; CecB is preferentially active in early pupae. A small peak of constitutive cecropin expression in early pupae appears to be caused by bacteria in the food. Cecropin A, the common product of the CecA1 and A2 genes, was identified in the hemolymph of immunized flies at a concentration of 25-50 microM, enough to kill all tested bacteria except Serratia, a Drosophila pathogen. A useful in vitro system to study the immune response has been found in Schneider's line 2 cells which respond to lipopolysaccharide and laminarin by cecropin expression.     

Cloning of cDNAs for Cecropins A and B,and Expression of the Genes in the Silkworm,Bombyx mori

cDNA clones coding cecropins A and B were isolated from a cDNA library constructed from the fat body of immunized Bombyx mori larvae. The cloned cDNAs had an open reading frame of 63 amino acids, indicating the primary translated peptides were processed to form mature cecropins of 35 amino acid residues. The homology in the coding regions of cecropins A and B was 73%.

In immunized fat body, the expression of both cecropin A and B genes reached the maximal level 5 h after the injection of soluble peptidoglycan, and the high level was maintained until 9 h after immunization. The cecropin A and B genes were expressed at high levels in fat body and hemocytes, at lower but significant levels in malpighian tube, slightly in midgut, and none in silk gland.