铜绿假单胞菌分泌蛋白Pec1抑制巨噬细胞自噬影响细菌清除的机制研究

Pseudomonas aeruginosa (PA) is prone to colonize and cause infection in the respiratory tract. Autophagy of macrophage (Mφ) plays an important role in the process of fighting infections. Pec1 protein is one of the virulence factors secreted by PA, however, its effect on the autophagy of Mφ has not been reported. Our previous studies have shown that the autophagy level decreased significantly, and intracellular bacterial count increased obviously after Pec1 stimulated Mφ. Furthermore, the phosphorylation of p38 MAPK was increased and the expressions of miR-155 and miR-199a-5p were significantly decreased after the stimulation of Mφ. In addition, expression of PI3K p85, Akt and mTOR phosphorylation of Mφ were remarkably increased after stimulation. Bioinformatics prediction shows that PI3K p85 is a possible target for these two miRNA. On this ground, we attempt to confirm the following mechanism via experiments in vivo and in vitro that Pec1 protein secreted by PA would activate the signal molecule p38 MAPK after Mφ infected by PA, thus down-regulating expression of miR-155 and miR-199a-5p, which would targeting-inhibit the subunit of degradation PI3K p85 and then activates PI3K/Akt/mTOR signaling pathway to inhibit Mφ autophagy, ultimately contributing to the survival of PA.

铜绿假单胞菌(PA)极易在呼吸道定植与感染，巨噬细胞(Mφ)自噬在抵抗PA感染的过程中发挥着重要作用。Pec1蛋白是PA分泌的毒力因子，有关其影响宿主Mφ自噬作用的相关研究未见文献报道。我们前期研究发现：Pec1蛋白刺激Mφ后，自噬水平显著下降，胞内细菌数显著增多；且Pec1刺激Mφ后p38 MAPK 磷酸化显著增高，miR-155及miR-199a-5p 显著下调；此外，刺激后Mφ的PI3K p85表达、Akt及mTOR磷酸化皆显著上升；生物信息学预测显示，PI3K p85亚基是miR-155及miR-199a-5p的可能靶标。据此，我们拟通过体内外实验证实如下机制：PA感染Mφ，其分泌的Pec1通过激活p38 MAPK信号分子，下调miR-155和miR-199a-5p表达，抑制对PI3K p85亚基的靶向降解，进而激活PI3K/Akt/mTOR通路抑制Mφ自噬，最终有利于PA的存活。

铜绿假单胞菌(PA)是广泛存在于环境中的条件致病菌，在免疫功能低下的个体中可引起致死性感染，亟须研究其毒力因子在感染过程中的作用以开发更有效的诊断和治疗方法。Pec 1蛋白是PA新的毒力因子，其在PA致病过程中的作用尚不清楚。在本项研究中，我们发现：Pec 1蛋白下调integrinβ3的表达，进而抑制miR-155的表达，导致PI3K、AKT、mTOR的磷酸化水平升高，PI3K-AKT-mTOR激活，抑制巨噬细胞(Mφ)自噬及对PA的清除。而Mφ转染integrin β3过表达质粒或miR-155 mimics、或PI3K抑制剂处理Mφ则观察到相反的结果。同样，体内实验证实，与PA标准株(PAO1)感染的大鼠相比，Pec 1敲除(PA△Pec 1)株导致的肺损伤程度较轻，肺部细菌负荷少，自噬水平较高。此外，PA肺部感染患者的痰液中可检测到Pec 1蛋白。本研究揭示了Pec 1蛋白在PA感染过程中通过抑制integrin β3的表达，进而抑制miR-155的表达，激活PI3K-AKT-mTOR，抑制Mφ自噬及对PA的清除。该研究为PA感染的快速诊断和治疗提供了新的思路。