Zhai Dongsheng, Wang Wenwen, Ye Zichen, Xue Ke, Chen Guo, Hu Sijun, Yan Zhao, Guo Yanhai, Wang Fang, Li Xubo, Xiang An, Li Xia, Lu Zifan, Wang Li
Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, Shaanxi, China.
State Key Laboratory of Cancer Biology, Department of Biopharmaceutics, Fourth Military Medical University, Xi'an, Shaanxi, China.
Cell Biosci. 2022 Sep 10;12(1):154. doi: 10.1186/s13578-022-00865-9.
Sepsis is a fatal condition commonly caused by Methicillin-resistant Staphylococcus aureus (MRSA) with a high death rate. Macrophages can protect the host from various microbial pathogens by recognizing and eliminating them. Earlier we found that Quaking (QKI), an RNA binding protein (RBP), was involved in differentiation and polarization of macrophages. However, the role of QKI in sepsis caused by pathogenic microbes, specifically MRSA, is unclear. This study aimed to investigate the role of QKI in regulation of host-pathogen interaction in MRSA-induced sepsis and explored the underlying mechanisms.
Transmission electron microscope and immunofluorescence were used to observe the autophagy level in macrophages. Real-time PCR and western blot were used to analyzed the expression of mRNA and protein respectively. The potential protein interaction was analyzed by iTRAQ mass spectrometry and Immunoprecipitation. RNA fluorescence in situ hybridization, dual-luciferase reporter assay and RNA immunoprecipitation were used to explore the mechanism of QKI regulating mRNA of PI3K-p110β.
The mRNA level of QKI was aberrantly decreased in monocytes and PBMCs of septic patients with the increasing level of plasma procalcitonin (PCT). Then the mice with myeloid specific knockout of QKI was challenged with MRSA or Cecal Ligation and Puncture (CLP). Mice in these two models displayed higher survival rates and lower bacterial loads. Mechanistically, QKI deletion promoted phagocytosis and autophagic degradation of MRSA via activating p110β (a member of Class IA phosphoinositide 3-kinases) mediated autophagic response. QKI expression in macrophages led to the sequestration of p110β in mRNA processing (P) bodies and translational repression. Upon infection, the direct interaction of RNF6, a RING-type E3 ligase, mediated QKI ubiquitination degradation and facilitated PI3K-p110β related autophagic removal of pathogen. The administration of nanoparticles with QKI specific siRNA significantly protected mice from MRSA infection.
This study disclosed the novel function of QKI in the P body mRNA regulation during infection. QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K-p110β dependent autophagy. It suggested that QKI may serve as a potential theranostic marker in MRSA-induced sepsis.
脓毒症是一种通常由耐甲氧西林金黄色葡萄球菌（MRSA）引起的致命疾病，死亡率很高。巨噬细胞可通过识别和清除各种微生物病原体来保护宿主。我们之前发现，RNA结合蛋白（RBP）震颤蛋白（QKI）参与巨噬细胞的分化和极化。然而，QKI在由病原微生物，特别是MRSA引起的脓毒症中的作用尚不清楚。本研究旨在探讨QKI在MRSA诱导的脓毒症中调节宿主-病原体相互作用的作用，并探索其潜在机制。
使用透射电子显微镜和免疫荧光观察巨噬细胞中的自噬水平。分别使用实时PCR和蛋白质印迹分析mRNA和蛋白质的表达。通过iTRAQ质谱和免疫沉淀分析潜在的蛋白质相互作用。使用RNA荧光原位杂交、双荧光素酶报告基因检测和RNA免疫沉淀来探索QKI调节PI3K-p110β mRNA的机制。
脓毒症患者的单核细胞和外周血单个核细胞（PBMC）中QKI的mRNA水平随着血浆降钙素原（PCT）水平的升高而异常降低。然后用MRSA或盲肠结扎穿孔（CLP）对髓系特异性敲除QKI的小鼠进行攻击。这两种模型中的小鼠均显示出较高的存活率和较低的细菌载量。机制上，QKI缺失通过激活I类A磷酸肌醇3激酶的成员p110β介导的自噬反应促进MRSA的吞噬和自噬降解。巨噬细胞中QKI的表达导致p110β在mRNA加工（P）小体中被隔离并导致翻译抑制。感染后，RING型E3连接酶RNF6的直接相互作用介导QKI泛素化降解，并促进PI3K-p110β相关的病原体自噬清除。给予携带QKI特异性siRNA的纳米颗粒可显著保护小鼠免受MRSA感染。
本研究揭示了QKI在感染期间P小体mRNA调节中的新功能。RNF6介导巨噬细胞中QKI降解通过增强PI3K-p110β依赖性自噬保护小鼠免受MRSA感染。这表明QKI可能作为MRSA诱导的脓毒症的潜在诊疗标志物。
