脂多糖和脂磷壁酸诱导果蝇抗菌肽基因表达的机理

Innate immune signaling pathways are conserved from insects to humans. For example, Drosophila melanogaster Toll-Sp?tzle and Imd (immune deficiency) pathways share similarities with mammalian Toll-like receptors (TLRs) and tumor necrosis factor (TNF)-alpha pathways, respectively. But Drosophila signaling pathways also differ from mammalian pathways in the recognition processes. Different mammalian TLRs directly recognize microbial components, such as lipopolysaccharide (LPS), peptidoglycan (PG), bacterial DNA and viral RNA molecules to activate signaling pathways. TLR4 recognizes LPS to activate the NF-κB pathway and simulate pro-inflammatory genes. But Drosophila Toll does not directly bind to any microbial components; instead, it binds to the cytokine-like ligand Sp?tzle to activate the Toll-Sp?tzle pathway and stimulate antimicrobial peptide (AMP) gene expression. In addition, in Drosophila, the Toll-Sp?tzle and Imd pathways are activated by Gram-negative and Gram-positive bacteria PGs, respectively, and LPS does not activate AMP genes in adult female flies. However, in some other insect species such as Manduca sexta and Bombyx mori, LPS and lipoteichoic acid (LTA), another major component of Gram-positive bacteria, can activate AMP gene expression. Thus, in this application, we want to ask the following three questions: (1) Can LPS and LTA, like PG, activate AMP gene expression in Drosophila? (2) If LPS and LTA can activate AMP genes, which signaling pathways, the Toll-Sp?tzle and/or Imd pathways, are involved in regulation of AMP gene expression? (3) In addition to Toll-1 and Sp?tzle -1, are any other Drosophila Tolls (Toll2-9) and Sp?tzle proteins (Sp?tzle2-6) involved in signaling pathways to regulate AMP gene expression? To answer these questions, wild-type Drosophila larvae, adult male and female flies will be fed diets containing or injected with ultra pure Gram-negative Escherichia coli LPS-K12 and PG-K12, Gram-positive Staphylococcus aureus LTA-SA and PG-SA as well as Gram-positive Bacillus subtilis LTA-BS and PG-BS, and larvae and adult flies will be collected at different time points post-infection for analysis of AMP gene expression to determine whether LPS and LTA can activate AMP genes. Then, mutant Drosophila flies defected in one of the key genes in the Toll-Sp?tzle or Imd pathway will be used for the feeding and injection experiments to determine which genes or pathways are involved in regulation of AMP genes activated by LPS or LTA. Finally, other Drosophila toll receptors and Sp?tzle proteins will be selected as candidate receptors and ligands to study their interactions by co-immunoprecipitation and protein pull-down assays, and promoter-luciferase assay will be used to test activation of AMP genes in Drosophila S2 cell lines co-expressing Toll receptor and Sp?tzle.

肽聚糖诱导果蝇抗菌肽表达的分子机制已研究的非常清楚，但脂多糖和脂磷壁酸这另外两种细菌体表主要成分是否能诱导果蝇抗菌肽的表达，目前还不清楚。在本项研究中，我们将选用果蝇这种模式昆虫为研究对象，用超纯的肽聚糖、脂多糖和脂磷壁酸免疫刺激果蝇，得到详细的抗菌肽表达模式，确认除肽聚糖外，脂多糖和脂磷壁酸是否能激活果蝇抗菌肽的表达；在此基础上，我们将通过突变体分析和RNAi分析，确认脂多糖和脂磷壁酸是通过哪个信号转导途径来激活抗菌肽表达的；此外我们还将研究果蝇Toll2-9及Spatzle 2-6蛋白间的相互作用和配对情况，分析这些Toll和Spatzle配对是否能激活抗菌肽基因的表达，并进一步确认这些Toll和Spatzle配对是否参与脂多糖或脂磷壁酸激活的Toll-Spatzle信号通路。研究结果将揭示脂多糖和脂磷壁酸诱导果蝇抗菌肽基因表达的分子机理，加深人们对昆虫先天性免疫机制的认识。

抗菌肽基因表达调控的机理一直是昆虫先天性免疫研究领域的热点，并取得了一些进展。但目前有些机理仍然不是很清楚，还需要开展更深入的研究。在本项目的资助下，我们选用果蝇这种模式昆虫为研究对象，较为系统地研究了细菌及细菌细胞壁成分（LPS等）激活果蝇抗菌肽基因表达的机理。研究结果显示：（1）喂食细菌（局部感染）也能激活果蝇抗菌肽基因的表达，并且是通过Toll 和 IMD信号通路来调控抗菌肽基因的表达的，初步揭示了喂食感染方式下果蝇抗菌肽基因表达的机理；另外，预先局部感染能够使果蝇对全身感染具有更高的抵抗力，表明果蝇可能通过协同利用局部免疫和全身免疫来使自己对病菌的感染具有更强的抵抗力；（2）除了PG外，细菌细胞壁的其他成分比如LPS也能诱导果蝇抗菌肽基因的表达，并且诱导模式在果蝇不同虫态及幼虫不同龄期是不一样的，表明LPS等细胞壁成分诱导果蝇抗菌肽表达的模式是非常复杂的；（3）除Toll-1和Spätzle-1外，其他Toll和Spätzle也能配对，并激活果蝇抗菌肽基因的表达；（4）三种NF-κB转录因子能通过形成同源和异源二聚体来调控果蝇抗菌肽基因的表达，表明这些NF-κB转录因子能协同调控果蝇抗菌肽的表达。这些研究结果揭示了果蝇抗菌肽基因表达调控的分子机理，加深了人们对昆虫先天性免疫机制的认识。本项目按期结题，完成了预定的研究目标，取得了预期的研究成果。