淋病奈瑟菌逃逸自噬杀伤机制的分子机理研究

Neisseria gonorrhoeae is a human-specific bacterial pathogen that colonizes the mucosal epithelial cell layers of the urogenital tract and causes the sexually transmitted disease gonorrhea. A critical step for N. gonorrhoeae to cause disseminated infections is its invasion of epithelial cells and transmigration to the sub-epithelial space. However, epithelial cells contain the autophagy pathway, which is an intracellular mechanism that is able to eliminate intracellular pathogens. It is currently unknown whether the autophagy pathway is also able to target and kill intracellular N. gonorrhoeae or whether N. gonorrhoeae has developed mechanisms to inhibit autophagy or escape autophagy-mediated degradation. Preliminary research for this project has already demonstrated that during the early stage of epithelial cell invasion N. gonorrhoeae was targeted by the autophagy pathway and sequestered into double-membrane autophagosomes. These early N. gonorrhoeae-containing autophagosomes were subsequently able to fuse with lysosomes thereby killing N. gonorrhoeae in the hitherto formed autolysosomes. However, a subpopulation of the intracellular gonococci was able to escape early autophagy-mediated killing. N. gonorrhoeae was subsequently able to inhibit this pathway, allowing intracellular survival and exocytosis. During this stage, N. gonorrhoeae activated mammalian target of rapamycin complex 1 (mTORC1), which resulted in repression of the canonical autophagy pathway. Furthermore, at this stage N. gonorrhoeae appeared to also inhibit autophagosome maturation into autolysosomes, which was independently from mTORC1 activation. Therefore, these data show that N. gonorrhoeae has multiple mechanisms to escape autophagy-mediated degradation during the later stage of epithelial cell invasion. Based on these preliminary results, the aim of this project is to define the specific interactions between N. gonorrhoeae and epithelial cell autophagy to elucidate the mechanisms by which autophagy is activated and able to target N. gonorrhoeae during the early stage of invasion and to elucidate the mechanism by which N. gonorrhoeae is able to escape autophagy-mediated killing during the later stage of epithelial cell invasion. Specific interactions with epithelial cells have already been described for several surface expressed proteins or virulence factors of N. gonorrhoeae and some of these interactions are feasible to have an impact on autophagy activation of modulation. Therefore, these specific interactions will be investigated in details to elucidate their mechanism and involvement in activation or modulation of autophagy during N. gonorrhoeae invasion of epithelial cells.

细菌病原体淋病奈瑟氏菌致病最关键的过程是其侵入并穿过上皮细胞进入上皮下间隙，从而造成弥散性感染。在上皮细胞中存在着一种可以清除病原微生物的机制——自噬。本课题组前期实验结果表明：在上皮细胞侵染的早期，淋球菌即可被自噬途径靶向识别并被隔离形成双层膜的自噬体。这些早期包含淋球菌的自噬体随后能够与溶酶体融合，从而在随后形成的自噬溶酶体中杀死淋球菌。但是，有一小部分胞内淋球菌能够逃避早期自噬介导的杀伤作用；随后淋球菌能够抑制自噬通路，从而使得病原菌能够在胞内存活和向胞外分泌。在淋球菌抑制自噬的过程中，淋球菌将通过激活mTOR复合物1以抑制经典自噬通路。此外，在此阶段淋球菌还可能抑制自噬体成熟及自噬溶酶体的形成。基于这些初步结果，本项目旨在深入探究淋球菌与上皮细胞自噬之间的具体相互作用，以阐明在侵染早期阶段自噬被激活并能够靶向淋球菌的机制以及淋球菌在侵染上皮细胞后期能够逃避自噬介导的杀伤作用的机制。

淋病奈瑟球菌是一种只能对人类造成感染的细菌性病原体，其可以定植于泌尿生殖道粘膜上皮，导致重要性传播疾病——淋病的发生。淋球菌能够侵入上皮细胞，但是进入胞内的细菌会被自噬途径所靶向。自噬是一种宿主细胞的防御机制，其形成具有双层膜结构的自噬泡来捕获入侵细菌，随后与溶酶体融合来使其降解。细胞内淋球菌和上皮细胞自噬系统之间的相互作用机制尚未被完全阐明。因此，本项目的总体目标是对淋球菌和自噬途径之间的相互作用进行详细分析，以阐明自噬对其胞内生存的影响。该项目使用庆大霉素保护试验、淋球菌缺失突变体、蛋白免疫印迹、共聚焦显微镜和免疫共沉淀实验，证明了在淋球菌入侵上皮细胞的早期阶段，其被自噬途径靶向，并被隔离到双膜自噬体，随后与溶酶体融合进而被破坏。在这一阶段，胞内的淋球菌被NOD1所识别，其随后招募IRGM和ATG16L1，以激活自噬并靶向早期和晚期内体中的淋球菌，随后使其在自噬体中被破坏。入侵的淋球菌通过外膜磷脂酶A逃避NOD1识别和被自噬靶向，从而在感染后期经胞吐形成弥散性感染。在此阶段，自噬体成熟和经典自噬途径被抑制。棕榈酸是细胞内含量最丰富脂肪酸，它是磷脂酶A的裂解产物。淋球菌对棕榈酸的适应有利于其感染上皮细胞和胞内生存。淋球菌侵染上皮细胞过程中也分泌大量外膜囊泡（OMV）。OMV能够运输到线粒体以诱导线粒体自噬，这是一种特殊的崭新的引起线粒体自噬发生的机制。线粒体自噬的发生依赖于外膜蛋白PorB，它破坏了线粒体膜电位从而引起线粒体被自噬系统降解。OMV诱导的线粒体自噬降低了线粒体生成活性氧的水平，这有利于淋球菌在胞内的生存。总体而言，该项目阐明了淋球菌与上皮细胞自噬系统之间相互作用的详细机制，也解释了淋球菌如何在细胞内存活并随后被胞吐的现象。这些实验结果与进展对淋病的研究是十分重要和关键的，因为这为干预粘膜上皮中淋球菌转胞吞过程提供了可靠的靶点，从而防止弥散性感染的发生。