人TLR9胞内段抗菌新功能域的确认及其在RAW264.7细胞处理内吞细菌中的调理作用

Monocyte/phagocytic cells employ toll-like receptor 9 (TLR9) as one of the key sensors to recognize pathogen molecular patterns, while its structure is still unclear. The intracellular domain of TLR9 is well known as the signal transduction subunit. Previously, we accidently found it has antibacterial effect.In the present study, we plan to isolate one or several antibacterial domains(ABD) as the candidates from TLR9 via bioinformatic analysis. The bacteria growth inhibition experiments will be carried out to screen the best ABD.Then full-lenth TLR9 and ABD-deletion mutant will be introduced into HEK293 cell respectively. The function to get rid of bacteria of different transgenic HEK293 cells will be detected by phagocytosis test, and then indentifying ABD at cell level. ABD will be finally overexpressed in RAW264.7 cell, phagocytosis test will also be carried out to detect the influence of ABD in engulfing and killing the bacteria. The different activated levels of TLR9 pathway will also be detected of both normal or tansgenic RAW264.7 cells. Finally, The oposonization of ABD to help Monocyte/phagocytic cells in getting rid of the endocytotic bacteria will be evaluated based on the previous results. This study will be helpful to recognize the structure and function of important pattern recognizing receptors, and also be helpful to discover new drug target.

Toll样受体9（Toll-like receptor 9，TLR9）是单核/吞噬细胞识别病原菌菌体成分的关键受体之一，其结构尚不清楚。胞内段是其信号转导功能域，前期研究偶然发现人TLR9的胞内段具有抗菌作用。本研究拟首先采用生物信息学分析人TLR9胞内段结构，获得一个或几个候选抗菌功能域（ABD），并采用细菌生长抑制实验筛选抗菌作用较强的片段；在HEK293细胞中分别转染人TLR9全长基因及其ABD缺失突变体，通过吞噬细菌实验比较野生型与突变体清除细菌能力，在细胞水平最终明确ABD；在RAW264.7细胞中过表达ABD，采用吞噬细菌实验观察ABD对细胞吞噬、杀灭和破坏细菌的影响及细胞TLR9通路的变化，探讨ABD对单核/吞噬细胞处理内吞细菌的调理作用。本研究将为拓展我们对重要模式识别受体TLR9的结构和功能的认识，发现新的药物作用靶点提供实验依据。

前期工作表明TLR9胞内段具有抗菌活性，本项目拟在此基础上确认该抗菌功能域（antibacterial domain，ABD）并探讨其对RAW264.7细胞处理内吞细菌中的调理作用。首先进一步确认TIR1段抗菌作用，在此基础上拆分TIR1为4段，分别合成多肽，采用二倍微孔稀释法和透射电镜法验证其抗菌作用，初步确认D3为ABD（880-908 AA）。其次，成功构建TLR9野生型(TLR9-WT)和D3缺失突变体(TLR9-ΔD3)的pcDNA3.0真核表达载体，并转染到HEK293T细胞中稳定表达，并通过western blot检测证实胞内TLR9受体的表达。吞噬细菌实验表明，转染TLR9-WT的细胞吞噬细菌能力显著提升，而转染TLR9-ΔD3的细胞吞噬能力较WT明显更弱，与普通细胞无差异，确认D3为ABD。采用可内化入胞的D3多肽直接孵育RAW264.7，观察其对细胞吞噬细菌能力的影响，发现D3可显著上调RAW264.7细胞吞噬、清除细菌能力。D3的作用与网格蛋白依赖的内化途径和清道夫受体无关，而与上调细胞自噬有关，自噬阻断后D3的作用消失。Western blot和共定位实验表明，D3的作用与激活MyD88-自噬途径有关，其分子机制为D3可上调MyD88表达与招募，从而诱导自噬。本项目成功将TLR9胞内段ABD定位到D3（880-908 AA），并明确其可促进RAW264.7细胞内化和清除细菌，该作用与激活MyD88-自噬途径有关。