Department of Bioresource Sciences, Andong National University, 388 Songchun, Andong, Republic of Korea.
Dev Comp Immunol. 2010 May;34(5):530-7. doi: 10.1016/j.dci.2009.12.013. Epub 2010 Jan 4.
Bacterial challenge enhances phospholipase A(2) (PLA(2)) activity, which in turn induces biosynthesis of various eicosanoids that mediate non-self recognition signal to immune effectors in insects. But, there is little information on how PLA(2) activity is controlled after the non-self recognition. A recent genome analysis of the red flour beetle, Tribolium castaneum, has annotated both Toll and Imd signal pathways that are presumably considered to specifically respond to different microbial infections to express specific antimicrobial peptides (AMPs). This study set up a hypothesis that PLA(2) activation is linked to Toll and Imd pathways in T. castaneum. Bacterial challenge to the larvae of T. castaneum induced expressions of Toll and Imd genes. Different AMP genes were induced in larvae infected with Gram-positive or -negative bacteria. RNA interference using double-stranded RNAs (dsRNAs) specific to different Toll and Imd genes showed differential inhibition of these AMP expressions, indicating that the Toll and Imd pathways play critical roles in the production of AMPs by specifically responding to various bacterial challenges in T. castaneum. These Toll and Imd immune signals are also linked to the activation of PLA(2) in T. castaneum. Activation of PLA(2) was significantly induced in response to bacterial challenge, but was inhibited by dsRNAs specific to different Toll and Imd genes. The activation of PLA(2) via Toll and Imd pathways could be explained by induction of PLA(2) gene expression because the dsRNA treatments of Toll and Imd genes suppressed the gene expression of PLA(2) in response to bacterial challenge. The functional links were further supported by an immunofluorescence assay of PLA(2) intracellular translocation. Upon bacterial challenge, hemocytes from control larvae showed intracellular translocation of their PLA(2)s near to cell membrane, but hemocytes from larvae treated with dsRNAs of the Toll and Imd genes did not show the translocation, at which the PLA(2)s appeared to be evenly spread in the cytoplasm. These results suggest that recognition of bacterial challenge initiates Toll and Imd pathways in T. castaneum, which subsequently induces the activation of immune-associated PLA(2)s as well as gene expression of various AMPs.
细菌挑战增强了磷脂酶 A（PLA（2））的活性，进而诱导各种类二十烷酸的生物合成，这些类二十烷酸介导昆虫中免疫效应器的非自身识别信号。但是，关于非自身识别后 PLA（2）活性如何控制的信息很少。红粉甲虫（Tribolium castaneum）的最近基因组分析注释了 Toll 和 Imd 信号通路，这两种信号通路被认为专门用于响应不同的微生物感染，以表达特定的抗菌肽（AMP）。本研究提出了一个假设，即 PLA（2）的激活与 T. castaneum 的 Toll 和 Imd 途径有关。细菌对 T. castaneum 幼虫的挑战诱导了 Toll 和 Imd 基因的表达。感染革兰氏阳性或阴性细菌的幼虫中诱导了不同的 AMP 基因。使用针对不同 Toll 和 Imd 基因的双链 RNA（dsRNA）进行 RNA 干扰显示，这些 AMP 表达的抑制存在差异，表明 Toll 和 Imd 途径在 T. castaneum 对各种细菌挑战的 AMP 产生中发挥关键作用。这些 Toll 和 Imd 免疫信号也与 T. castaneum 中 PLA（2）的激活有关。细菌挑战显著诱导了 PLA（2）的激活，但被针对不同 Toll 和 Imd 基因的 dsRNA 特异性抑制。通过 Toll 和 Imd 途径激活 PLA（2）可以通过诱导 PLA（2）基因表达来解释，因为 Toll 和 Imd 基因的 dsRNA 处理抑制了细菌挑战时 PLA（2）基因的表达。免疫荧光分析 PLA（2）细胞内转位进一步支持了功能联系。在细菌挑战后，对照幼虫的血细胞显示其 PLA（2）在细胞膜附近的细胞内转位，但用 Toll 和 Imd 基因的 dsRNA 处理的幼虫的血细胞没有显示出转位，此时 PLA（2）似乎均匀分布在细胞质中。这些结果表明，细菌挑战的识别在 T. castaneum 中启动了 Toll 和 Imd 途径，随后诱导了与免疫相关的 PLA（2）的激活以及各种 AMP 的基因表达。
