United States Department of Agriculture, Agriculture Research Service, National Center for Cool and Cold Water Aquaculture, 11861 Leetown Road, Kearneysville, WV 25430, USA.
Fish Shellfish Immunol. 2010 May-Jun;28(5-6):918-26. doi: 10.1016/j.fsi.2010.02.002. Epub 2010 Feb 11.
Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam(2)CSK(4)) and triacylated lipoprotein (Pam(3)CSK(4)). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.
先天免疫途径的诱导对于早期抗微生物防御至关重要，但对于鱼类如何识别微生物分子并激活这些途径的理解有限。在哺乳动物中，Toll 样受体（TLR）1 和 2 形成异二聚体，参与识别微生物来源的肽聚糖和脂蛋白。在此，我们鉴定并描述了虹鳟（Oncorhynchus mykiss）TLR1 基因的直系同源物及其 mRNA 表达。使用 DNA 测序和遗传连锁分析，从虹鳟细菌人工染色体（BAC）文库中鉴定并描述了两个 TLR1 基因座。全长 cDNA 克隆和对四个 BAC 的直接测序显示，14 号染色体上存在一个完整的 omTLR1 开放阅读框（ORF），25 号染色体上存在第二个包含 TLR1 假基因的基因座。重复的鱼类基因座与其他鱼类基因组具有保守的同线性，超出了 TLR1 基因序列。omTLR1 基因包含一个类似于所有其他描述的 TLR1 基因的单个大编码外显子，但与其他硬骨鱼不同，它还具有 5'UTR 外显子和大编码外显子之前的内含子。omTLR1 ORF 预测编码一个 808 个氨基酸的蛋白质，与 Fugu TLR1 具有 69%的相似性，并具有保守的预测亮氨酸丰富重复（LRR）模式。系统发育分析将 omTLR1 与其他鱼类 TLR1 基因聚集成一个与禽类 TLR1 和哺乳动物 TLR1、6 和 10 分开的分支。虹鳟前肾白细胞中的 omTLR1 表达水平不受人类 TLR2/6 和 TLR2/1 激动剂二酰化脂蛋白（Pam(2)CSK(4)）和三酰化脂蛋白（Pam(3)CSK(4)）的影响。然而，由于 TLR6 和 10 基因在硬骨鱼基因组中的缺失以及其他鱼类 TLR1 mRNA 在 LPS 和细菌感染下的上调，我们假设 omTLR1 在抗微生物免疫中具有重要作用。因此，鉴定虹鳟 TLR2 直系同源物并开发测量配体结合和下游信号转导的测定法对于未来阐明 omTLR1 功能至关重要。
