肺炎克雷伯菌调控肺泡Ⅱ型上皮细胞自噬的机制研究
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摘要
目的探讨肺泡Ⅱ型上皮细胞自噬体与肺炎克雷伯菌相互作用的分子机制。方法建立体外肺炎克雷伯菌(K.pneumoniae)感染A549细胞模型,分为感染组(Pneu)、自噬抑制组(Pneu+3-MA)、自噬诱导组(Pneu+rapa)、阴性对照组(ctrl),每组3个样本;通过免疫荧光染色与western-blot实验检测肺炎克雷伯菌不同时间点及不同浓度感染后细胞中自噬相关蛋白LC3Ⅱ的表达变化及分布情况;进一步采用自噬抑制剂3-Methyladenine (3-MA)与激动剂雷帕霉素(Rapamycin)干预细胞,观察K.pneumoniae感染A549细胞后自噬水平的变化。结果与未感染肺炎克雷伯菌的对照组相比,共聚焦显微镜观察感染组细胞LC3自噬体发生明显改变(P<0.05),western-blot检测感染组细胞LC3Ⅱ蛋白表达上升(P<0.05),且LC3-Ⅱ/LC3-Ⅰ升高(P<0.05),表明肺炎克雷伯菌能够促进细胞自噬,随着细菌感染细胞比例(MOI)的增大,细胞自噬随之增多,随着时间的延长,肺炎克雷伯菌对细胞的入侵能力减弱(自噬增加),LC3Ⅱ蛋白的表达逐渐增加。自噬抑制剂和激动剂处理细胞后,免疫荧光和western-blot检测结果显示,感染组、自噬诱导组的LC3Ⅱ蛋白高于对照组(P<0.05),采用3-MA处理的感染组LC3Ⅱ蛋白低于感染组(P<0.05),表明3-MA能够抑制细胞的自噬,Rapamycin可以促进细胞的自噬。结论自噬在肺泡Ⅱ型上皮细胞抵抗肺炎克雷伯菌过程中发挥着非常重要的作用。
OBJECTIVE To investigate the molecular mechanism of the interaction between autophagosome of alveolar type Ⅱ epithelial and Klebsiella pneumoniae. METHODS Models of A549 cell infected with K. pneumoniae was established in vitro, and divided into infected group(Pneu), autophagy inhibition group(Pneu+3-MA), autoph-agy induction group(Pneu+rapa) and negative control group(ctrl), with 3 samples in each group. Western-blot and immunofluorescence staining were used to detect the expression of LC3Ⅱ and the distribution of an autophagy-associated protein LC3 at different time points after treatment with K. pneumoniae at different concentrations. After intervention with the autophagy inhibitor 3-methyladenine(3-MA) and the agonist rapamycin, the level of autopha-gy in A549 cells after K. pneumoniae infection was further examined. RESULTS Compared with the control group without K. pneumoniae infection, the expression of LC3Ⅱ in the in-fected group was significantly changed as observed by confocal microscopy(P<0.05). Western blot analysis showed that the expression of LC3Ⅱ protein in the infected group was significantly increased(P<0.05), and LC3-Ⅱ/LC3-Ⅰ was significantly increased(P<0.05), indicating that K. pneumoniae could promote autophagy, and as the proportion of bacterially infected cells(MOI) increased, autophagy increased. With the prolongation of time, the ability of K. pneumoniae to invade cells decreased(increased autophagy), and the expression of LC3Ⅱ protein gradually increased. After treatment with autophagy inhibitors and agonists, immunofluorescence and western-blot assays showed that the levels of LC3Ⅱ protein in the bacterial infection group and the autophagy induction group were significantly higher than that in the control group(P<0.05). The level of LC3Ⅱ protein in the group was significantly lower than that in the infected group(P<0.05), indicating that 3-MA inhibited autophagy and rapamycin promoted autophagy. CONCLUSION Autophagy plays an important role in the resistance of alveolar type Ⅱ epithelial cells to K. pneumoniae.
OBJECTIVE To investigate the molecular mechanism of the interaction between autophagosome of alveolar type Ⅱ epithelial and Klebsiella pneumoniae. METHODS Models of A549 cell infected with K. pneumoniae was established in vitro, and divided into infected group(Pneu), autophagy inhibition group(Pneu+3-MA), autoph-agy induction group(Pneu+rapa) and negative control group(ctrl), with 3 samples in each group. Western-blot and immunofluorescence staining were used to detect the expression of LC3Ⅱ and the distribution of an autophagy-associated protein LC3 at different time points after treatment with K. pneumoniae at different concentrations. After intervention with the autophagy inhibitor 3-methyladenine(3-MA) and the agonist rapamycin, the level of autopha-gy in A549 cells after K. pneumoniae infection was further examined. RESULTS Compared with the control group without K. pneumoniae infection, the expression of LC3Ⅱ in the in-fected group was significantly changed as observed by confocal microscopy(P<0.05). Western blot analysis showed that the expression of LC3Ⅱ protein in the infected group was significantly increased(P<0.05), and LC3-Ⅱ/LC3-Ⅰ was significantly increased(P<0.05), indicating that K. pneumoniae could promote autophagy, and as the proportion of bacterially infected cells(MOI) increased, autophagy increased. With the prolongation of time, the ability of K. pneumoniae to invade cells decreased(increased autophagy), and the expression of LC3Ⅱ protein gradually increased. After treatment with autophagy inhibitors and agonists, immunofluorescence and western-blot assays showed that the levels of LC3Ⅱ protein in the bacterial infection group and the autophagy induction group were significantly higher than that in the control group(P<0.05). The level of LC3Ⅱ protein in the group was significantly lower than that in the infected group(P<0.05), indicating that 3-MA inhibited autophagy and rapamycin promoted autophagy. CONCLUSION Autophagy plays an important role in the resistance of alveolar type Ⅱ epithelial cells to K. pneumoniae.
引文
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[2] Hoxha A,Karki T,Giambi C,et al.Attributable mortality of carbapenem-resistant Klebsiella pneumoniae infections in a prospective matched cohort study in Italy,2012-2013[J].J Hosp Infect,2016,92(1):61-66.
[3] Zhang X,Gu B,Mei Y,et al.Increasing resistance rate to carbapenem among blood culture isolates of Klebsiella pneumoniae,Acinetobacter baumannii and Pseudomonas aeruginosa in a university-affiliated hospital in China,2004-2011.[J].J Antibiot (Tokyo),2015,68(2):115-120.
[4] Fraenkel-Wandel Y,Raveh-Brawer D,Wiener-Well Y,et al.Mortality due to blaKPC Klebsiella pneumoniae bacteraemia[J].J Antimicrob Chemother,2016,71(4):1083-1087.
[5] Shi Z,Zhao H,Li G,et al.Molecular characteristics of carbapenem-resistant Enterobacter cloacae in Ningxia Province,China[J].Front Microbiol,2017,8:94.
[6] 师志云,赵慧铮,李彩云,等.烧伤科患者肠杆菌科细菌耐药性与基因型分布[J].中华医院感染学杂志,2016,26(12):2660-2663.
[7] 李刚,赵慧铮,贾伟.7315株肠杆菌科细菌的临床分布与耐药性分析[J].国际检验医学杂志,2015,36(6):786-787.
[8] 刘香花,师志云,李刚,等.宁夏地区某医院耐碳青霉烯酶肠杆菌科细菌临床分布及耐药基因的分析[J].中华医院感染学杂志,2018,28(5):659-662.
[9] Nakagawa I,Amano A,Mizushima N,et al.Autophagy defends cells against invading group A Streptococcus [J].Science,2004,306(5698):1037-1040.
[10] Crotzer VL,Blum JS.Autophagy and intracellular surveillance:modulating MHC class II antigen presentation with stress[J].Proc Natl Acad Sci U S A,2005,102(22):7779-7780.
[11] Stegemann-Koniszewski S,Jeron A,Gereke M,et al.Alveolar type II epithelial cells contribute to the anti-influenza A virus response in the lung by integrating pathogen- and microenvironment-derived signals[J].mBio,2016,7(3):e00276-16.
[12] 朱任媛,张小江,徐英春,等.2012年中国CHINET无菌体液中分离的细菌构成和耐药性监测[J].中国感染与化疗杂志,2014,14(6):482-487.
[13] Liu Y,Wang JY,Jiang W.An increasing prominent disease of Klebsiella pneumoniae liver abscess:etiology,diagnosis,and treatment[J].Gastroenterol Res Pract,2013:258514.
[14] Baraniak A,Izdebski R,Fiett J,et al.NDM-producing Enterobacteriaceae in Poland,2012-14:inter-regional outbreak of Klebsiella pneumoniae ST11 and sporadic cases[J].J Antimicrob Chemother,2016,71(1):85-91.
[15] CDC.Biggest Threats and Data[EB/OL](2013).http://www.cdc.gov/drugresistance/threat-report-2013/[23/4/2013].
[16] World Health Organization.Antimicrobial resistance:global report on surveillance 2014[EB/OL](2014).http://www.who.int/drugresistance/documents/surveillancereport/en/[2014-06-01].
[17] Noad J,von der Malsburg A,Pathe C,et al.LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-κB[J].Nat Microbiol,2017,2:17063.
[18] Negroni A,Colantoni E,Vitali R,et al.NOD2 induces autophagy to control AIEC bacteria infectiveness in intestinal epithelial cells[J].Inflamm Res,2016,65(10):803-813.
[19] Yuan K,Huang C,Fox J,et al.Autophagy plays an essential role in the clearance of Pseudomonas aeruginosa by alveolar macrophages[J].J Cell Sci,2012,125(Pt 2):507-515.
[20] Cemma M,Kim PK,Brumell JH.The ubiquitin-binding adaptor proteins p62/SQSTM1 and NDP52 are recruited independently to bacteria-associated microdomains to target Salmonella to the autophagy pathway [J].Autophagy,2011,7(3),341-345.
[21] Guo F,Zhang H,Chen C,et al.Autophagy favors Brucella melitensis survival in infected macrophages [J].Cell Mol Biol Lett,2012,17(2):249-257.
[22] Romagnoli A,Etna MP,Giacomini E,et al.ESX-1 dependent impairment of autophagic flux by mycobacterium tuberculosis in human dendritic cells[J].Autophagy,2012,8(9):1357-1370.
[23] Sato K,Akaki T,Shimizu T,et al.Invasion and intracellular growth of Mycobacterium tuberculosis and Mycobacterium avium complex adapted to intramacrophagic environment within macrophages and type II alveolar epithelial cells[J].Kekkaku,2001,76(2):53-57.
[24] Zhao YX,Li G,Yu RJ.[The effect of retinoic acid on C3 and factor B secretion of human alveolar type II epithelial cells induced with cytokines].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2005,21(4):442-444.
[25] Yamagata T,Yamagata Y,Nishimoto T,et al.The regulation of amiloride-sensitive epithelial sodium channels by tumor necrosis factor-alpha in injured lungs and alveolar type II cells[J].Respir Physiol Neurobiol,2009,166(1):16-23.
[26] Marsh LM,Cakarova L,Kwapiszewska G,et al.Surface expression of CD74 by type II alveolar epithelial cells:a potential mechanism for macrophage migration inhibitory factor-induced epithelial repair[J].Am J Physiol Lung Cell Mol Physiol,2009,296(3):L442-L452.
[27] Aberle H,Butz S,Stappert J,et al.Assembly of the cadherin-catenin complex in vitro with recombinant proteins [J].J Cell Sci,1994,107 (Pt 12):3655-3663.
[28] Hippenstiel S,Opitz B,Schmeck B,et al.Lung epithelium as a sentinel and effector system in pneumcnia-molecular mechanisms of pathogen recognition and signal transduction [J].Respir Res,2006,7:97.
[29] Zhao YX,Li G,Yu RJ.The effect of retinoic acid on C3 and factor B secretion of human alveolar type II epithelial cells induced with cytokines[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2005,21(4):442-444.
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