Full sequence and characterization of two insect defensins: immune peptides from the mosquitoAedes aegypti (original) (raw)

Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedes aegypti and a defensin D from Aedes albopictus

Insect Molecular Biology, 1999

An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7±10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A dierential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we de®ned as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the ®nal amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12± 36 h after induction.

Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti

Insect Molecular Biology, 2004

Defensin is the predominant inducible immune peptide in Aedes aegypti. In spite of its activity against Grampositive bacteria in vitro , defensin expression is detected in mosquitoes inoculated with Gram-positive or negative bacteria, or with filarial worms. Defensin transcription and expression are dependent upon bacterial dose; however, translation is inconsistent with transcription because peptide is detectable only in mosquitoes inoculated with large doses. In vitro translation assays provide further evidence for posttranscriptional regulation of defensin. Clearance assays show that a majority of bacteria are cleared before defensin is detected. In gene silencing experiments, no significant difference in mortality was observed between defensin-deficient and control mosquitoes after bacteria inoculation. These studies suggest that defensin may have an alternative function in mosquito immunity.

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

Biochemical and Biophysical Research Communications, 1998

We have identified a 35-amino acid antibiotic cecropin capitata (8). secreted by an established mosquito cell line. C7-10 cells Cecropins are small (É4 kDa) peptides containing from the vector mosquito, Aedes albopictus, were incu-35-39 amino acids, in which a high proportion of basic bated with heat-killed Escherichia coli, and materials amino acids at the N-terminus confers a net positive secreted into the cell culture supernatant were recovcharge, while the C-terminus is rich in hydrophobic ered by acid precipitation. Following batch elution from residues. Structural analyses indicate that the N-and Sep-Pak C18 cartridges and further purification by re-C-terminal portions of the peptide form a-helical strucverse phase high performance liquid chromatography tures, which are separated by a gly-pro bend in the (RP-HPLC) a predominant peak of antibacterial activity well-studied cecropin A from H. cecropia and cecropin was characterized by mass spectrometry, amino acid B2 from B. mori (9). The insect cecropins show broadcomposition analysis, and Edman degradation, yielding spectrum activity against Gram-negative and Gramthe sequence GGLKKLGKKLEGVGKRVFKASEKALPVpositive bacteria, but differ from other antibacterial AVGIKALG. Unlike other cecropins, the peptide was not amphipathic a-helical peptides, such as magainins and amidated at the C-terminus. Aedes albopictus Cecropin dermaseptins from frog skin, in that they do not lyse A (AalCecA) is the first cecropin to be described from a mosquito vector of human disease. Consistent with the erythrocytes (1, 9). classification of mosquitoes among the Dipteran subor-Of particular interest to mosquito biologists and parder Nematocera, AalCecA shares only 36% amino acid asitologists was the observation that cecropins and synidentity with cecropins from Drosophila melanogaster thetic derivatives thereof have activity against pathoand other Cyclorrhaphid flies, whose mature cecropins genic protozoa, including agents that cause malaria share 80% to 100% amino acid identity. ᭧ 1998 Academic Press and trypanosomiasis (10, 11, 12). The possibilities for Key Words: insect; mosquito; immunity factors; subverting the endogenous immune response in moscecropins. quitoes and other vector arthropods to interrupt pathogen transmission (13) have stimulated efforts to identify immunity factors from these species, resulting thus far in the molecular characterization of antibacterial Upon bacterial infection, insects synthesize a battery defensins from various mosquitoes (14), but mosquito of antibacterial proteins and peptides, which have been cecropins have not yet been resolved. In this paper, we grouped into families based on structural similarities. describe the purification and amino acid sequence of a Among the first such peptides to be sequenced were 35-residue cecropin secreted by an established cell line cecropins A and B from the moth, Hyalophora cecropia from the vector mosquito, Aedes albopictus. (1). More recently, cecropin sequences have been deduced from cDNA clones or directly determined from MATERIALS AND METHODS several lepidoptera, including Manduca sexta (2), Antheraea pernyi (3), and Bombyx mori (4), as well as Cell line and sample preparation. Ae. albopictus C7-10 cells were from several flies classified among the higher Diptera, maintained in Eagle's medium supplemented with 5% fetal bovine including the flesh fly, Sarcophaga peregrina (5), the serum (E-5 medium) as described previously (15). Cells were plated pomace flies Drosophila melanogaster (6) and Drosophin 100-mm tisssue culture plates (50-100 plates), and grown to 50 to 80% confluency in E-5 medium. To facilitate protein purification, cells were treated in serum-free (E-0) medium with heat-killed Escherichia coli NM 539 (Ç1000 bacteria per cell) as described by Hernan-1

The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes

Insect Biochemistry and Molecular Biology, 2001

A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 µM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.

Insect antimicrobial peptides and their applications

Applied microbiology and biotechnology, 2014

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 .

Characterization of New Defensin Antimicrobial Peptides and Their Expression in Bed Bugs in Response to Bacterial Ingestion and Injection

International Journal of Molecular Sciences

Common bed bugs, Cimex lectularius, can carry, but do not transmit, pathogens to the vertebrate hosts on which they feed. Some components of the innate immune system of bed bugs, such as antimicrobial peptides (AMPs), eliminate the pathogens. Here, we determined the molecular characteristics, structural properties, and phylogenetic relatedness of two new defensins (CL-defensin1 (XP_024085718.1), CL-defensin2 (XP_014240919.1)), and two new defensin isoforms (CL-defensin3a (XP_014240918.1), CL-defensin3b (XP_024083729.1)). The complete amino acid sequences of CL-defensin1, CL-defensin2, CL-defensin3a, and CL-defensin3b are strongly conserved, with only minor differences in their signal and pro-peptide regions. We used a combination of comparative transcriptomics and real-time quantitative PCR to evaluate the expression of these defensins in the midguts and the rest of the body of insects that had been injected with bacteria or had ingested blood containing the Gram-positive (Gr+) bact...

CHARACTERISTICS OF ANTIMICROBIAL PEPTIDES ORIGINATED FROM INVERTEBRATES INCLUDING INSECT

Most AMPs discovered in invertebrates were found in arthropods including insect, and antibacterial activity was discovered by specifically high frequency against Gram positive bacteria. The length of peptides discovered the highest amount in 31~40 peptides, and it maintained comparatively higher levels in 11~50 peptides. The secondary structure of peptides showed the highest value in alpha-helix, but very lower amounts in beta-sheet and coil form. As net charge was ranged from +1 to +7, it was observed to have a net charge slightly higher than general AMPs. Aliphatic index was observed to have the largest amount between 70 and 80, which more than half of the peptides were found to form a stable structure. Therefore, it is suggested that AMPs found in invertebrates have characteristics similar to AMPs discovered in all the organisms.