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.     

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.     

cDNA cloning and gene expression of cecropin D, an antibacterial protein in the silkworm, Bombyx mori

We have isolated a cDNA clone encoding a cecropin D precursor from the fat body of Bombyx mori larvae immunized with bacteria by means of differential display. The cDNA contains 298 bp with a coding region of 183 bp for 61 amino acids plus a termination codon (TAG), a 5′-untranslated region of 36 bp, and a 3′-untranslated region of 79 bp including the poly(A) tail. There is a polyadenylation signal sequence of AATAAA at position 266, 43 nucleotides downstream from the termination codon TAG. The homology of the deduced amino acids is greater to the cecropin D precursor from Hyalophora cecropia (67% identity) than to the precursors of cecropins A and B from B. mori (49% to both). Northern blotting analyses reveal that the gene expression of cecropin D is detectable by 4 h after the bacterial injection and reaches the maximal level at 24 h. That high level is maintained up to 48 h post-immunization. Additionally, the gene is expressed mainly in the fat body and slightly in hemocytes, but it is undetectable in other tissues such as the midgut, the Malpighian tubule and silk gland.     

Drosophila cecropin as an antifungal agent

The effects of Drosophila and Hyalophora cecropins were tested against different fungi, both insect pathogens and fungi from the normal environment of Drosophila. The fungi were generally found to be as susceptible to the cecropins as most bacteria, the only exception being the insect pathogen Beauveria bassiana which is completely resistant. This is also the only fungus tested which is virulent to Drosophila, giving 100% lethality within 5 days after injection. Lethal concentrations of cecropins against other fungi tested ranged between 0.4 and 4 microM. Andropin is less fungicidal than the cecropins, and Drosophila cecropin A is somewhat more potent than cecropin B. Even dense cultures of Saccharomyces cerevisiae can be cleared by micromolar concentrations of cecropin, whereas Geotrichum candidum is unaffected by cecropin when tested in a dense culture.     

Molecular evolution of the cecropin multigene family in silkworm Bombyx mori

Cecropins constitute one of the largest and most potent immune protein families found in insect species with diversified numbers and features. In view of the large number of cecropin proteins existing with much sequence variations among them, an overview of the multigene cecropin family in silkworm Bombyx mori was attempted in this study. Cecropin encodes an inducible 64 residue anti-bacterial peptide and was clustered into two groups; first group viz. A and second group including B, D, E and Enbocin. Cecropin A consisted of two sub-groups located on chromosome number 6 of B.mori genome. Cecropin B consisted of six sub-groups, cecropin D and E of one each and Enbocin of two. The second sub-group formed in tandem array of multigene family locus over a length of 78.62 kb on chromosome number 26 in B.mori genome and was organized in positive as well as opposite orientation. The results indicated that cecropin B genes were organized in a close cluster with the intergenic sequence ranging from 1366 bp to 23526 bp. Interestingly a distantly related cecropin E was also located within the cecropin B multigene locus. Similarly distant members like cecropin D and Enbocin were also located in the 3' region of cecropin B locus. The maximum intergenic region of 23526 bp observed between Cecropin D and Enbocin indicates that the two genes were distantly evolved. The phylogenetic analysis clearly indicates a positive correlation between the clusters and physical location on the chromosome, as the length of the intergenic region plays a major role to create newer cecropin families. EST database analysis suggests that most of the cecropin A members were expressed in the microbial fat body while, the cecropin B was equally expressed in fat body and other target tissues. The signal peptides were conserved in all the twelve paralogous gene sequences.     

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.     

斜纹夜蛾两个天蚕素D基因的克隆及序列分析

天蚕素是昆虫抵御病菌入侵的一类抗菌肽家族。根据斜纹夜蛾Spodoptera litura天蚕素B基因设计特异引物,通过PCR扩增得到2个新的天蚕素基因部分序列,分别命名为cecD1和cecD2（GenBank登录号分别为EF555567和EF555568）。2个基因编码同一个天蚕素D蛋白,该蛋白的成熟肽与天蚕素B存在2个氨基酸残基差异。序列分析发现cecD1和cecD2中分别包含568 bp和377 bp的内含子序列,它们有相同的5′和3′拼接位点,A+T含量分别为59.7%和69.8%,符合大多数真核生物内含子高A+T含量的特征。     

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.     

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.     

CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae.

Cecropins are antibacterial peptides, induced in insects in response to bacterial infections. In Drosophila, three cecropin genes have previously been characterized, CecA1, CecA2, and CecB, in a dense cluster at 99E on the third chromosome. From the same locus, we now describe a fourth member of the cecropin gene family, CecC, which is mainly expressed at the early pupal stage. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth.     

Molecular population genetics of inducible antibacterial peptide genes in Drosophila melanogaster

Insects respond to septic infection in part by producing a suite of antimicrobial peptides that may be subject to host-pathogen coevolutionary dynamics. In order to infer population genetic forces acting on Drosophila antibacterial peptide genes, we examine global properties of polymorphism and divergence in the Drosophila melanogaster defensin, drosocin, metchnikowin, attacin C, diptericin A, and cecropin A, B, and C genes. As a functional class, antibacterial peptides exhibit low levels of interspecific amino acid divergence. There are multiple amino acid polymorphisms segregating within D. melanogaster, however, a high proportion of which change the charge or polarity of the variable residue. These polymorphisms are particularly prevalent in processed signal and propeptide domains. We find that models of coevolutionary "arms races" and selectively maintained hypervariability do not adequately describe the population dynamics of mature antibacterial peptides in D. melanogaster, but that a highly significant excess of high-frequency derived polymorphisms coupled with substantial intralocus linkage disequilibrium suggests that positive selection may act on antibacterial peptide genes. Some attributes of the data may be consistent with a simple demographic model of population founding followed by expansion, but departures from the equilibrium null tend to be more pronounced in the peptide genes than at other loci around the genome.     

cDNA cloning and antibacterial activities of cecropin D-like peptides from Agrius convolvuli

We have characterized full-length cDNAs encoding two isoforms of agriusin, cecropin D-like antibacterial peptide, present in the hemolymph of the immunized Agrius convolvuli larvae. The cloned cDNAs of agriusins 1 and 2 contain 331 and 329 bp, respectively. The nucleotide sequencing of cDNAs showed that they encode 62 amino acids, whose mature portion was deduced to consist of 38 amino acid residues with over 94% sequence identity. In the sequence homology search, mature agriusin 1 showed over 86 and 71% amino acid sequence homology with bactericidin 4 from Manduca sexta and cecropin D from Hyalophora cecropia, respectively. Since it was demonstrated from the deduced amino acid sequences that the C-terminal residues of agriusins are followed by a Gly residue, two types of synthetic agriusin 1 (syn-agriusin 1 amide and acid) were prepared to verify if natural agriusin 1 is C-terminally amidated. From acid-urea PAGE and reversed phase HPLC profiles to compare two synthetic peptides, we could confirm that the C-terminal amino acid residue of natural agriusin 1, like several cecropins so far identified, is amidated. Finally, our antibacterial assay performed with two syn-agriusins 1 revealed that there is little difference between antibacterial activities of both peptides against Gram-positive and Gram-negative bacteria.     

Characterization and cDNA cloning of hinnavin II, a cecropin family antibacterial peptide from the cabbage butterfly, Artogeia rapae

Hinnavins, together with lysozymes, are the main types of antibacterial peptides/proteins previously isolated from the larval haemolymph of the cabbage butterfly, Artogeia rapae as part of the humoral immune response to a bacterial invasion. One of these antibacterial peptides, named hinnavin II, was purified and characterized after cDNA cloning. The purified hinnavin II was more active against Gram negative than against Gram positive bacteria. Hinnavin II also showed a powerful synergistic effect on the inhibition of bacterial growth with purified lysozyme. The cDNA has a total length of 186 bp with a 114 coding region. The deduced protein sequence contains 38 amino acids with a coding capacity of 4142.8 Da. The result of a multiple sequence alignment and phylogenetic analysis with Clustal W indicated that mature hinnavin II showed an approximately 78.9% amino acid sequence identity with cecropin A and originated from a group containing mostly lepidopteran cecropins.     

Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide

The anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic alpha-helix) and its derivative, cecropin B3 (CB3; having two hydrophobic alpha-helices) on the membrane of Ags cancer cells. Application of 10-60 microM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimicrobial peptides at the single cancer cell level.