Identification of immunity-related genes distinctly regulated by Manduca sexta Spӓtzle-1/2 and Escherichia coli peptidoglycan

IF 3.2 2区 农林科学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Insect Biochemistry and Molecular Biology Pub Date : 2024-03-27 DOI:10.1016/j.ibmb.2024.104108
Zelong Miao, Chao Xiong, Yang Wang, Tisheng Shan, Haobo Jiang
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Abstract

The immune system of Manduca sexta has been well studied to understand molecular mechanisms of insect antimicrobial responses. While evidence supports the existence of major immune signaling pathways in this species, it is unclear how induced production of defense proteins is specifically regulated by the Toll and Imd pathways. Our previous studies suggested that diaminopimelic acid-type peptidoglycans (DAP-PG) from Gram-negative and some Gram-positive bacteria, more than Lys-type peptidoglycans (Lys-PG) from other Gram-positive bacteria, triggers both pathways through membrane-bound receptors orthologous to Drosophila Toll and PGRP-LC. In this study, we produced M. sexta proSpätzle-1 and proSpätzle-2 in Sf9 cells, identified their processing enzymes, and used prophenoloxidase activating protease-3 to activate the cytokine precursors. After Spätzle-1 and -2 were isolated from the reaction mixtures, we separately injected the purified cytokines into larval hemocoel to induce gene transcription in fat body through the Toll pathway solely. On the other hand, we treated a M. sexta cell line with E. coli DAP-PG to only induce the Imd pathway and target gene expression. RNA-Seq analysis of the fat body and cultured cells collected at 0, 6, and 24 h after treatment indicated that expression of diapausin-4, -10, -12, -13, cecropin-2, -4, -5, attacin-5, -11, and lebocin D is up-regulated predominantly via Toll signaling, whereas transcription of cecropin-6, gloverin, lysozyme-1, and gallerimycin-2 is mostly induced by DAP-PG via Imd signaling. Other antimicrobial peptides are expressed in response to both pathways. Transcripts of most Toll-specific genes (e.g., lebocin D) peaked at 6 h, contrasting the gradual increase and plateauing of drosomycin mRNA level at 24−48 h in Drosophila. We also used T (oll)-I (md) ratios to estimate relative contributions of the two pathways to transcriptional regulation of other components of the immune system. The differences in pathway specificity and time course of transcriptional regulation call for further investigations in M. sexta and other insects.

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鉴定受曼陀罗Spӓtzle-1/2和大肠杆菌肽聚糖独特调控的免疫相关基因
为了了解昆虫抗微生物反应的分子机制,对肉曼陀罗(Manduca sexta)的免疫系统进行了深入研究。虽然有证据支持该物种存在主要的免疫信号通路,但目前还不清楚Toll和Imd通路是如何具体调控防御蛋白的诱导产生的。我们以前的研究表明,来自革兰氏阴性菌和一些革兰氏阳性菌的二氨基亚氨酰基肽聚糖(DAP-PG)比来自其他革兰氏阳性菌的赖氨酸型肽聚糖(Lys-PG)更能通过与果蝇的 Toll 和 PGRP-LC 同源的膜结合受体触发这两种途径。在这项研究中,我们在 Sf9 细胞中产生了 M. sexta proSpätzle-1 和 proSpätzle-2,鉴定了它们的加工酶,并使用丙醇氧化酶激活蛋白酶-3 来激活细胞因子前体。从反应混合物中分离出Spätzle-1和-2后,我们分别将纯化的细胞因子注射到幼虫血球中,使其仅通过Toll途径诱导脂肪体中的基因转录。另一方面,我们用大肠杆菌 DAP-PG 处理 M. sexta 细胞系,只诱导 Imd 通路和靶基因的表达。在处理后 0、6 和 24 小时收集的脂肪体和培养细胞的 RNA-Seq 分析表明,DAP-PG 主要通过 Toll 信号途径上调 diapausin-4、-10、-12、-13、cecropin-2、-4、-5、attacin-5、-11 和 lebocin D 的表达,而 cecropin-6、gloverin、溶菌酶-1 和 gallerimycin-2 的转录则主要通过 Imd 信号途径诱导。其他抗菌肽在两种途径下都会表达。大多数 Toll 特异性基因(如 lebocin D)的转录本在 6 小时内达到峰值,而果蝇的 drosomycin mRNA 水平在 24-48 小时内逐渐增加并趋于平稳。我们还利用T(ll)-I(md)比率来估算两种途径对免疫系统其他成分转录调控的相对贡献。在M. sexta和其他昆虫中,途径特异性和转录调控时间过程的差异需要进一步研究。
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来源期刊
CiteScore
7.40
自引率
5.30%
发文量
105
审稿时长
40 days
期刊介绍: This international journal publishes original contributions and mini-reviews in the fields of insect biochemistry and insect molecular biology. Main areas of interest are neurochemistry, hormone and pheromone biochemistry, enzymes and metabolism, hormone action and gene regulation, gene characterization and structure, pharmacology, immunology and cell and tissue culture. Papers on the biochemistry and molecular biology of other groups of arthropods are published if of general interest to the readership. Technique papers will be considered for publication if they significantly advance the field of insect biochemistry and molecular biology in the opinion of the Editors and Editorial Board.
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Identification of a gene promoter active in Lucilia sericata larval salivary glands using a rapid transient expression assay. In vivo RNAi screening identifies multiple deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila. JAK and STAT5B mediate olfactory response of migratory locusts to their own volatiles. Functional redundancy of the three insulin receptors of cockroaches. Editorial Board
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