Keestra A Marijke, de Zoete Marcel R, van Aubel Rémon A M H, van Putten Jos P M
Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
Mol Immunol. 2008 Mar;45(5):1298-307. doi: 10.1016/j.molimm.2007.09.013. Epub 2007 Oct 26.
Mammalian Toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and activates the innate immune system. The avian TLR repertoire exhibits considerable functional diversity compared to mammalian TLRs and evidence of a functional TLR5 in the avian species is lacking. In the present study we cloned and successfully expressed chicken TLR5 (chTLR5) in HeLa cells, as indicated by laser confocal microscopy. Infection of chTLR5 transfected cells with Salmonella enterica serovar Enteritidis activated NF-kappaB in a dose- and flagellin-dependent fashion. Similar NF-kappaB activation was observed with recombinant bacterial flagellin. Targeted mutagenesis of the proline residue at position 737 in the chTLR5-TIR domain was detrimental to chTLR5 function, confirming that the observed effects were conferred via chTLR5 and the MyD88 signaling pathway. Comparison of human, mouse and chicken TLR5 activation by flagellin of S. enterica serovar Typhimurium revealed that chTLR5 consistently yielded stronger responses than human but not mouse TLR5. This species-specific reactivity was not observed with flagellin of serovar Enteritidis. The species-specific TLR5 response was nullified after targeted mutagenesis of a single amino acid (Q89A) in serovar Typhimurium flagellin, while L415A and N100A substitutions had no effect. These results show that chickens express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. The finding that single amino acid substitutions in bacterial flagellin can alter the species-specific TLR5 response may influence the host range and susceptibility of infection.
哺乳动物的Toll样受体5（TLR5）可识别多种细菌的鞭毛蛋白并激活先天免疫系统。与哺乳动物的TLR相比，禽类的TLR库表现出相当大的功能多样性，且缺乏禽类功能性TLR5的证据。在本研究中，我们在HeLa细胞中克隆并成功表达了鸡TLR5（chTLR5），激光共聚焦显微镜观察结果表明了这一点。用肠炎沙门氏菌肠炎血清型感染chTLR5转染的细胞，可剂量依赖性和鞭毛蛋白依赖性方式激活核因子κB（NF-κB）。用重组细菌鞭毛蛋白也观察到了类似的NF-κB激活。对chTLR5-TIR结构域中第737位脯氨酸残基进行靶向诱变对chTLR5功能有害，证实观察到的效应是通过chTLR5和髓样分化因子88（MyD88）信号通路介导的。比较鼠伤寒沙门氏菌鞭毛蛋白对人、小鼠和鸡TLR5的激活作用，发现chTLR5产生的反应始终比人TLR5更强，但比小鼠TLR5弱。肠炎血清型鞭毛蛋白未观察到这种物种特异性反应。在鼠伤寒沙门氏菌鞭毛蛋白中对单个氨基酸（Q89A）进行靶向诱变后，物种特异性TLR5反应消失，而L415A和N100A替换没有影响。这些结果表明，鸡表达功能性TLR5，尽管与人类TLR5相比，其鞭毛蛋白感应特性不同。细菌鞭毛蛋白中的单个氨基酸替换可改变物种特异性TLR5反应这一发现可能会影响宿主范围和感染易感性。
