Toll-related receptors and the control of antimicrobial peptide expression in Drosophila

Insects defend themselves against infectious microorganisms by synthesizing potent antimicrobial peptides. Drosophila has appeared in recent years as a favorable model to study this innate host defense. A genetic analysis of the regulation of the antifungal peptide drosomycin has demonstrated a key role for the transmembrane receptor Toll, which prompted the search for mammalian homologs. Two of these, Toll-like receptor (TLR)2 and TLR4, recently were shown to play a critical role in innate immunity against bacteria. Here we describe six additional Toll-related genes (Toll-3 to Toll-8) in Drosophila in addition to 18-wheeler. Two of these genes, Toll-3 and Toll-4, are expressed at a low level. Toll-6, -7, and -8, on the other hand, are expressed at high levels during embryogenesis and molting, suggesting that, like Toll and 18w, they perform developmental functions. Finally, Toll-5 is expressed only in larvae and adults. By using chimeric constructs, we have tested the capacity of the signaling Toll/IL-1R homology domains of these receptors to activate antimicrobial peptide promoters and found that only Toll and Toll-5 can activate the drosomycin promoter in transfected cells, thus demonstrating specificity at the level of the Toll/IL-1R homology domain. In contrast, none of these constructs activated antibacterial peptide promoters, suggesting that Toll-related receptors are not involved in the regulation of antibacterial peptide expression. This result was independently confirmed by the demonstration that a dominant-negative version of the kinase Pelle can block induction of drosomycin by the cytokine Spaetzle, but does not affect induction of the antibacterial peptide attacin by lipopolysaccharide.

昆虫通过合成强效抗菌肽抵御传染性微生物。果蝇（Drosophila）近年来已成为研究此类固有宿主防御机制的理想模型。针对抗真菌肽果蝇素（drosomycin）的调控开展遗传分析后，证实跨膜受体Toll发挥关键调控作用，这一发现推动了哺乳动物同源受体的搜寻工作。其中两类同源物——Toll样受体（Toll-like receptor，TLR）2与TLR4，近期被证实于抗细菌固有免疫中发挥关键作用。本研究额外报道了果蝇中除18-wheeler之外的6个Toll相关基因（Toll-3至Toll-8）。其中Toll-3与Toll-4的表达水平较低；Toll-6、-7和-8则在胚胎发生与蜕皮过程中呈高表达，提示其与Toll和18w（18-wheeler的缩写）类似，承担发育相关功能。而Toll-5仅在幼虫与成虫体内表达。通过构建嵌合融合体，本研究检测了这些受体的Toll/白介素1受体同源结构域（Toll/IL-1R homology domain）激活抗菌肽启动子的能力，结果显示仅Toll与Toll-5可在转染细胞中激活果蝇素启动子，从而证实了Toll/IL-1R同源结构域层面的信号特异性。与之相对，所有上述构建体均无法激活抗菌肽启动子，提示Toll相关受体并不参与抗菌肽表达的调控。该实验结果得到独立验证：激酶Pelle的显性负突变体可阻断细胞因子Spaetzle介导的果蝇素诱导，但不会影响脂多糖（lipopolysaccharide）诱导的抗菌肽攻击素（attacin）的表达。