The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes (original) (raw)
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The malaria vector mosquito Anopheles gambiae expresses a suite of larval-specific defensin genes
Insect Molecular Biology, 2008
cDNAs of Anopheles gambiae Defensin 2 (AgDef2), Defensin 3 (AgDef3) and Defensin 4 (AgDef4), identified in the genome sequence, have been characterized and their expression profiles investigated. In contrast to both typical defensins and insect antimicrobial peptides generally, the newly identified defensins were not upregulated with acute-phase kinetics following immune challenge in insects or cell culture. However, mRNA abundance of AgDef2, AgDef3 and AgDef4 increased significantly during the larval stages. Promoter analysis of all three genes failed to identify putative immune response elements previously identified in other mosquito defensin genes. As previous studies failed to identify these larval-specific defensins, it seems likely that further antimicrobial peptide genes with nontypical expression profiles will be identified as more genome sequences become available.
Acta Tropica, 2008
Manipulating the endogenous immune responses of the mosquito such as temporal and spatial expression of antimicrobial peptides may help in the development of a refractory mosquito, unable to transmit malaria. In mosquito several small antimicrobial peptides are activated locally in the midgut and salivary glands upon Plasmodium infection. Anopheles stephensi, the major urban malaria vector in India, has been considered as an important insect model to study vector-parasite interactions; however, so far no reports are available on the antimicrobial peptides from this mosquito species. In the present study, we report identification and molecular characterization of a novel cDNA encoding defensin like peptide, isolated from the salivary gland subtractive hybridization cDNA library of mosquito A. stephensi. Defensin cDNA is 396 base pair long, bearing an open reading frame of 96 amino acids. Deduced amino acid sequence of A. stephensi defensin (Astp def) contains a signal peptide sequence of 24 amino acids followed by 32-amino acids long putative propeptide domain and a 40-amino acid mature peptide domain carrying 23-amino acid long consensuses sequence signature of insect defensin. Mature peptide of Astp def carries six conserved cysteine residues, with a predicted molecular weight of 4.20 kDa, and isoelectric point of 8.30, characteristic features of cationic defensins. Amino acid sequence similarity and phylogenetic analysis indicated a higher variation in the pre-propeptide region, as compared to the mature defensin peptide, assuring the presence of finely tuned immune responses to counter pathogens.
Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene
EMBO Reports, 2002
Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector-parasite interactions. Here, we developed a simple in vivo doublestranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.
Proceedings of the Royal Society of London. Series B: Biological Sciences, 1995
We report the complete amino acid sequence and biological activity of two immune peptides, from the yellow fever mosquito Aedes aegypti, that are induced in response to infection. Both peptides display biological activity against the Gram positive microbe Micrococcus luteus and substantial sequence homology to insect defensins, small heat-stable, antibiotic peptides previously described from several non-vector insects. These mosquito peptides, designated Ae. aegypti defensins A and B, are isoforms. Defensin B is the most abundant antibacterial peptide in this species whereas defensin A is much less abundant and carries two amino acid substitutions compared to defensin B, making it more basic in character. Apparent convergence between isoforms from Ae. aegypti and the fleshfly Phormia terranovae is discussed. The synergistic activity previously described between Ae. aegypti immune haemolymph and lysozyme is not caused by these peptides because synergy occurred only at concentrations far outside the physiological range seen in Ae. aegypti.
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.
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.
Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae
Insect Molecular Biology, 2000
Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.
Genomic organization and immune regulation of the defensin gene from the mosquito, Anopheles gambiae
Insect Molecular Biology, 2000
The defensin gene from the mosquito, Anopheles gambiae , is present as a single copy per haploid genome. Two exons, encoding a 102 residue preprodefensin, are separated by a 105 bp intron bounded by consensus splice sites. The upstream regulatory sequence includes a TATA box, arthropod initiator and numerous motifs homologous to insect and mammalian immune response elements. This promoter is capable of upregulation by immune challenge in cultured cells and activity is further stimulated by Gambif 1, a mosquito Rel protein known to translocate to the nucleus and bind NF-κ κ κ κ B sites in target promoters. Activity is inhibited by p50, a mammalian Rel protein that competitively binds NF-κ κ κ κ B sites, and virtually abolished by p40, an avian I κ κ κ κ B protein that inhibits nuclear translocation.
Gambicin: A novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae
Proceedings of the National Academy of Sciences, 2001
A novel mosquito antimicrobial peptide, gambicin, and the corresponding gene were isolated in parallel through differential display-PCR, an expressed sequence tag (EST) project, and characterization of an antimicrobial activity in a mosquito cell line by reverse-phase chromatography. The 616-bp gambicin ORF encodes an 81-residue protein that is processed and secreted as a 61-aa mature peptide containing eight cysteines engaged in four disulfide bridges. Gambicin lacks sequence homology with other known proteins. Like other Anopheles gambiae antimicrobial peptide genes, gambicin is induced by natural or experimental infection in the midgut, fatbody, and hemocyte-like cell lines. Within the midgut, gambicin is predominantly expressed in the anterior part.