Yamamoto Masahiro, Standley Daron M, Takashima Seiji, Saiga Hiroyuki, Okuyama Megumi, Kayama Hisako, Kubo Emi, Ito Hiroshi, Takaura Mutsumi, Matsuda Tadashi, Soldati-Favre Dominique, Takeda Kiyoshi
Department of Microbiology and Immunology, Graduate School of Medicine, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
J Exp Med. 2009 Nov 23;206(12):2747-60. doi: 10.1084/jem.20091703. Epub 2009 Nov 9.
Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.
刚地弓形虫感染以依赖Stat3的方式下调宿主固有免疫反应，如促炎细胞因子的产生。最近一种正向遗传学方法表明，II型菌株由于一种高度多态的寄生虫来源激酶ROP16存在潜在缺陷，无法抑制免疫反应。我们通过反向遗传学产生了ROP16缺陷的I型寄生虫，发现寄生虫诱导的Stat3激活存在严重缺陷，最终导致受感染巨噬细胞中白细胞介素（IL）-6和IL-12 p40的产生增加。此外，在哺乳动物细胞中过表达ROP16而非ROP18会导致Stat3磷酸化以及Stat3依赖性启动子的强烈激活。此外，激酶失活的ROP16无法激活Stat3。I型和II型ROP16的比较表明，激酶结构域中的单个氨基酸取代决定了Stat3激活方面的菌株差异。此外，ROP16与Stat3结合并直接诱导该转录因子的磷酸化。这些结果正式确立了ROP16在寄生虫诱导的Stat3激活中的必需且直接的作用，以及单个氨基酸替换在II型ROP16功能中的重要性。
