BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 249, 410–415 (1998) ARTICLE NO. RC989150 Peptide Sequence of an Antibiotic Cecropin from the Vector Mosquito, Aedes albopictus Dongxu Sun,* Eric D. Eccleston,† and Ann M. Fallon* ,1 *Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St. Paul, Minnesota 55108; and †MicroChemical Facility, Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455 Received July 7, 1998 ila virilis (7), and the Mediterranean fruit fly, Ceratitis 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 recov- charge, 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 struc- verse 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 broad- composition analysis, and Edman degradation, yielding spectrum activity against Gram-negative and Gram- the sequence GGLKKLGKKLEGVGKRVFKASEKALPV- positive 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 par- der Nematocera, AalCecA shares only 36% amino acid asitologists was the observation that cecropins and syn- identity with cecropins from Drosophila melanogaster thetic derivatives thereof have activity against patho- and 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 mos- cecropins. quitoes and other vector arthropods to interrupt patho- gen transmission (13) have stimulated efforts to iden- tify 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 de- duced from cDNA clones or directly determined from MATERIALS AND METHODS several lepidoptera, including Manduca sexta (2), An- theraea 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 Drosoph- in 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 Esch- erichia coli NM 539 (Ç1000 bacteria per cell) as described by Hernan- 1 Corresponding Author. Fax: 612-625-5299. E-mail: fallo002@ maroon.tc.umn.edu. dez et al. (16). After 16 h, cells were removed by centrifugation, 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 410