Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta.
Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta.
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DOI：
10.1016/j.dci.2016.03.006
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发表时间：
2016-08
影响因子：
2.9
通讯作者：
Kanost MR
中科院分区：
文献类型：
--
作者：
Al Souhail Q;Hiromasa Y;Rahnamaeian M;Giraldo MC;Takahashi D;Valent B;Vilcinskas A;Kanost MR
昆虫分泌抗菌肽，作为先天免疫反应的一部分。大多数昆虫来源的抗菌肽具有抗菌活性，但不具有抗真菌活性。我们从鳞翅目昆虫的血淋巴中鉴定了一种抗真菌的多肽，Diaposin-1。用体积排阻层析法从预先注射酿酒酵母的幼虫血淋巴血浆中分离到Diaposin-1。用SDS-PAGE和MALDI-TOF MS/MS对含有抗酿酒酵母活性的部分进行分析，发现含有45个残基的多肽，该多肽是由转录组和基因组数据库中鉴定的序列编码的。从脂肪体RNA制备的cDNA中克隆了水泡素-1的全长基因。Diaposin-1是Diaposin多肽家族的成员之一，该家族包括已知具有抗真菌活性的成员。Sexta基因组包含14个与diaposin-1高度相似的基因，每个基因含有6个保守的Cys残基。Diaposin-1在大肠杆菌中以重组蛋白的形式表达。纯化的重组Diaposin-1对酿酒酵母有一定的抑制作用，IC_(50)为12μM，但未检测到抑菌活性。一些植物病原菌的孢子经水泡素-1处理后，萌发管卷曲或菌丝生长减慢或分枝。注射酵母菌或黄微球菌后，脂肪体中Diaposin-1mRNA水平显著升高。此外，在化蛹前的游荡幼虫阶段，滞育蛋白-1mRNA在中肠和脂肪体中的表达增加，这表明该基因对发育有调节作用。我们的结果表明，在六分枝杆菌感染后，Diaposin-1的合成是抗真菌先天免疫反应的一部分。
Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC50 of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta.