Porphyromonas gingivalis is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to P. gingivalis, but P. gingivalis escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of P. gingivalis, we analyzed the TLR2 interactome induced following P. gingivalis infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The P. gingivalis infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to P. gingivalis or to the synthetic TLR2/1 ligand Pam3Cys-Ser-(Lys)4 (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to P. gingivalis and reduced inflammatory cytokine production. We found that P. gingivalis drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9’s role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by P. gingivalis, leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and P. gingivalis immune escape in macrophages downstream of TLR2 sensing.

中文翻译：

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牙龈卟啉单胞菌诱导的 TLR2 相互作用组分析揭示与 PARP9 的关联

牙龈卟啉单胞菌是一种与牙周病密切相关的革兰氏阴性厌氧细菌。Toll 样受体 2 (TLR2) 对于宿主对牙龈卟啉单胞菌的反应是不可或缺的，但牙龈卟啉单胞菌通过磷酸肌醇 3-激酶 (PI3K) 的 TLR2 依赖性激活来逃避免疫清除。为了探究牙龈卟啉单胞菌依赖于 TLR2 的逃逸途径，我们分析了牙龈卟啉单胞菌感染或合成脂肽 TLR2/1 激动剂对过表达 TLR2 的人巨噬细胞激活后诱导的 TLR2 相互作用组。通过交联稳定相互作用的蛋白质，然后进行免疫沉淀并通过质谱分析。总共回收了 792 个蛋白质，网络分析能够绘制基线和感染反应中的 TLR2 相互作用组图。牙龈卟啉单胞菌感染诱导的 TLR2 相互作用组包括聚（ADP-核糖）聚合酶家族成员单 ADP-核糖基转移酶蛋白 9 (PARP9) 和 PARP9 复合物的其他成员（DTX3L 和 NMI）。PARP9 及其复合体成员在暴露于牙龈卟啉单胞菌或合成 TLR2/1 配体 Pam 的巨噬细胞中高度上调3半胱氨酸-丝氨酸-(赖氨酸)4（PAM）。与其在病毒诱导的干扰素产生中的已知作用一致，PARP9 敲低可阻断针对牙龈卟啉单胞菌的 I 型干扰素 (IFN-I) 产生，并减少炎症细胞因子的产生。我们发现牙龈卟啉单胞菌通过TLR2-PARP9驱动信号转导器和转录激活（STAT）1（S727）磷酸化，解释了PARP9在诱导TLR2下游IFN-I中的作用。此外，PARP9 敲除减少了牙龈卟啉单胞菌对 PI3K 的激活，从而提高了巨噬细胞的杀菌活性。总之，PARP9 是一种新型的 TLR2 相互作用伴侣，能够在 TLR2 传感下游的巨噬细胞中诱导 IFN-I 并实现牙龈卟啉单胞菌免疫逃逸。