Supplementary Material for: A<b><i> Staphylococcus aureus</i></b> TIR Domain Protein Virulence Factor Blocks TLR2-Mediated NF-κB Signaling

Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen <i>Staphylococcus aureus</i> produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in <i>S. aureus</i> MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to<i> S. aureus</i>. The effects on NF-κB pathway were confirmed using <i>S. aureus</i> MSSA476 wild type, an isogenic mutant MSSA476Δ<i>tirS</i>, and complemented MSSA476Δ<i>tirS +</i>pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new <i>S. aureus</i> virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo.Toll样受体（Toll-like receptors, TLRs）是介导宿主防御反应诱导的关键分子，其信号转导依赖于含有Toll/白细胞介素-1受体（Toll/interleukin-1 receptor, TIR）结构域的接头蛋白。病原菌金黄色葡萄球菌（Staphylococcus aureus）可产生多种天然免疫逃逸分子，以干扰宿主天然免疫应答。通过数据库搜索分析，研究人员在金黄色葡萄球菌菌株MSSA476中发现了一段编码人类TIR结构域同源蛋白的基因，并将其命名为葡萄球菌TIR结构域蛋白（staphylococcal TIR domain protein, TirS）。在人类胚胎肾细胞、巨噬细胞及角质形成细胞系中异位表达TirS，可干扰TLR2介导的信号转导通路，包括MyD88、TIRAP、NF-κB及/或丝裂原活化蛋白激酶（mitogen-activated protein kinase）通路。此外，TirS的存在可降低宿主细胞针对金黄色葡萄球菌应答所分泌的细胞因子MCP-1与G-CSF的水平。研究人员利用可将细菌与宿主细胞物理分隔的Transwell系统，分别采用金黄色葡萄球菌MSSA476野生型菌株、同基因敲除突变株MSSA476ΔtirS以及互补菌株MSSA476ΔtirS +pTirS，证实了TirS对NF-κB通路的调控效应。最后，在系统性小鼠感染模型中，感染后4天，TirS可促进细菌在多个器官内的定植。本研究结果揭示了一种新型金黄色葡萄球菌毒力因子，该因子可在体外干扰病原相关分子模式（pathogen-associated molecular pattern, PAMP）诱导的天然免疫信号转导，并在体内促进细菌存活。