Lan Jiang-Feng, Zhao Li-Juan, Wei Shun, Wang Yuan, Lin Li, Li Xin-Cang
Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, Hubei, 430070, China.
East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of East China Sea and Oceanic Fishery Resources Exploitation, Ministry of Agriculture, Shanghai, 200090, China.
Fish Shellfish Immunol. 2016 Nov;58:59-66. doi: 10.1016/j.fsi.2016.09.007. Epub 2016 Sep 10.
Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine transcription factor PcDorsal, into the nucleus participating in the immune defense against Gram-negative bacteria.
果蝇Toll蛋白和哺乳动物Toll样受体（TLRs）是一类在进化上保守的免疫受体家族，在抵御入侵病原体的一线防御中发挥关键作用。激活转录因子4（ATF4）是ATF/CREB转录因子家族的成员，是参与TLR信号传导和其他生理过程的重要因子。然而，在甲壳类动物中，ATF4同源物是否参与TLR信号传导仍不清楚。在本研究中，我们从克氏原螯虾中鉴定出一个Toll同源物PcToll2和一个新的ATF4同源物PcATF4，并分析了PcATF4在PcToll2信号传导中可能的调控活性。PcToll2的完整cDNA序列长4175 bp，包含一个2820 bp的开放阅读框，编码一个939个氨基酸的蛋白质，PcATF4的cDNA序列长2027 bp，开放阅读框为1296 bp，编码一个431个氨基酸的蛋白质。PcToll2与人TLR4具有高度同源性，它们被归为一个簇。此外，PcToll2与其他具有潜在抗菌活性的虾类TLRs关系密切。PcToll2在血细胞、心脏和鳃中高表达，而PcATF4主要分布在鳃中。在用副溶血性弧菌攻击后，PcToll2和PcATF4以及抗菌肽ALF1和ALF2在血细胞中显著上调，并且PcATF4易位到细胞核中。在PcToll2沉默并用弧菌攻击后，PcATF4向细胞核的易位受到抑制，ALF1和ALF2的表达降低，但PcDorsal和PcSTAT的表达不受影响。此外，在PcATF4敲低并在有无弧菌攻击后，ALF1和ALF2的表达也降低，而PcToll2的表达上调。这些结果表明，PcToll2可能通过促进PcATF4而非常规转录因子PcDorsal进入细胞核来调节ALF1和ALF2的表达，从而参与对革兰氏阴性菌的免疫防御。
