| 其他摘要 | 胞内病原微生物感染可以引起人类和动物很多严重的感染性疾病。胞内细菌感染如肺结核、伤寒、兔热病、Q热等，严重威胁着人类的健康，造成巨大的医疗负担。例如，每年接近有900万人感染由结核分支杆菌（Mycobacterium tuberculosis）引起的肺结核（13%的人同时感染HIV），大约有140万人死亡。其他的病原微生物如恶性疟原虫（Plasmodium falciparum），单核细胞增生李斯特菌（Listeria monocytogenes），脑膜炎双球菌（Neisseria meningitidi），类鼻疽假单胞杆菌（Pseudomonas pseudomallei），沙眼衣原体（Chlamydia trachomatis）以及一些病毒，同样也造成了相当大的发病率和死亡率。然而宿主细胞自身如何抵御胞内病原微生物的确切机制还不清楚。实验室前期在大蹼铃蟾皮肤分泌物中鉴定出一种孔道形成蛋白βγ-CAT，它可以寡聚化并在机体细胞膜上形成孔道。它由两个亚基：α亚基和β亚基组成。α亚基由两个与眼睛晶状体βγ-晶状体蛋白结构相似的βγ-晶状体蛋白结构域（βγ-crystalline）和一个与产气单胞菌溶（Aerolysins）结构相似的Aerolysin结构域组成，β亚基是一个含3个三叶因子结构域的三叶因子。实验室前期工作发现，能通过募集免疫细胞，激活炎症小体，引发IL-1β的成熟与释放，抵御胞外细菌。本研究中，我们分离培养了大蹼铃蟾腹腔细胞、脾细胞、胃肠细胞、肠上皮细胞、成纤维细胞，检测了孔道形成蛋白βγ-CAT在细胞上的表达情况与存在形式，利用胞内细菌L. monocytogenes建立了大蹼铃蟾成纤维细胞感染模型和腹腔感染模型。通过大蹼铃蟾腹腔感染胞内细菌L. monocytogenes模型，发现内源性孔道形成蛋白βγ-CAT能保护大蹼铃蟾抵御胞内细菌L. monocytogenes，提高个体存活率，降低感染细菌数。通过抑菌实验，我们发现βγ-CAT并不能直接杀死胞内细菌L. monocytogenes。体外实验证明，βγ-CAT这种内源性孔道形成蛋白可由胞内细菌L. monocytogenes刺激诱导产生。胞内细菌刺激细胞后，孔道形成蛋白βγ-CAT mRNA表达水平上升，形成寡聚体的程度也会增加，并会分泌到细胞外；包含βγ-CAT的囊泡也会增加，并能与包含L. monocytogenes的囊泡共定位。孔道形成蛋白βγ-CAT刺激细胞后，自噬水平上升，形成自噬小体。 本研究工作首次揭示了孔道形成蛋白βγ-CAT 在大蹼铃蟾细胞水平上的组成性表达，它能帮助宿主抵御胞内细菌L. monocytogenes，建立了成纤维细胞感 染模型，初步探索了βγ-CAT在帮助宿主抵御胞内细菌L. monocytogenes的可能作用机制，即可能通过细胞自主免疫——自噬，来清除宿主已感染的胞内病原细菌。; Intracellular pathogens can cause serious infectious diseases in humans and animals. Intracellular bacteria infections such as tuberculosis, typhoid fever, tularemia, Q fever and others are serious threat to human health and cause huge health burden. For example, each year there are nearly 900 million patient with tuberculosis (13% co-infection with HIV), caused about 1.4 million deaths. Other pathogens such as Plasmodium falciparum, Listeria monocytogenes, Neisseria meningitidi, as well as some viruses also cause considerable morbidity and mortality. However, the exact mechanism by which host cells defend themselves against intracellular pathogens is largely unclear.In previous studies of our laboratory, we have identified a pore-forming protein (PFP) complex βγ-CAT in Bombina maxima skin secretions. It can oligomerizes and forms a pore on cell membranes. βγ-CAT is a complex of a bacterial aerolysin-type pore-forming toxin-like protein (Bm-ALP1) and trefoil factor (Bm-TFF3) in frog Bombina maxima. We have also found that βγ-CAT can protect frog against extracellular bacterial infection by recruiting immune cells, subsequently activating the inflammasome, at last inducing the maturation and release of IL-1β.In this study, we want to investigate the function and the possible mechanisms of βγ-CAT which helps the host fight against intracellular pathogens. Firstly, we successfully isolated and cultured Bombina maxima cells. Then we detected the PFP complex βγ-CAT expression of the cell and found that βγ-CAT was endogenously expressed in kinds of frog cells. Thirdly, the Bombina maxima fibroblast infection model in vitro and abdominal infection model in vivo were established by using L. monocytogenes. In vivo studies, we found that the endogenous PFP complex βγ-CAT can protect Bombina maxima against intracellular bacteria L. monocytogenes, the survival of L. monocytogenes-infected Bombina maxima significantly improved after treatment with βγ-CAT. Meanwhile, the survival L. monocytogenes in the peritoneal cells and splenocytes of infected-frog markedly decreased 24 h after bacterial infection. Interestingly, βγ-CAT did not show any direct antibacterial activity to L. monocytogenes. The stimulation of intracellular bacteria L. monocytogenes can induce the secrection of the endogenous PFP complex βγ-CAT in vitro. βγ-CAT mRNA expression levels in frog peritoneal cells was significantly increased after L. monocytogenes infection. In addition, the secretion and oligomerization of βγ-CAT were also improved after L. monocytogenes challenge. The results of immunostaining showed that βγ-CAT was a vacuole-like pattern in frog cells after endocytosis. The number of βγ-CAT-containing vacuoles were increased and colocalized with the L. monocytogenes-containing vacuole. Furthermore, βγ-CAT can improve the conversion of LC3-I to LC3-II and the formation of autophagosome.In conclusion, this work firstly reveals that the PFP protein complex βγ-CAT, an aerolysin-Like PFP from vertebrate, is constitutively expressed in kinds of frog cells and help the host eradicate intracellular bacteria L. monocytogenes. Importantly, our findings demonstrated for the first time that host pore-forming proteins play a pivotal role in host innate immunity against intracellular pathogens, which may be at least partially explained by autophagy activation. |
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