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细菌与自噬的抗争——死亡或重生

本站小编 Free考研考试/2021-12-26

王招弟, 丁红雷
西南大学 动物科技学院 兽医传染病学实验室,重庆 400715
收稿日期:2019-12-08;接收日期:2020-02-21;网络出版时间:2020-04-28
基金项目:兽医生物技术国家重点实验室开放课题基金(No. SKLVBF201905),中央高校基本科研业务费专项资金(No. XDJK2020B012),重庆市技术创新与应用发展专项(No. cstc2019jscx-msxmX0402)资助

摘要:自噬是一种高度保守的细胞内成分的降解过程, 不仅维持细胞的代谢稳定, 还与机体对抗各种病原菌感染有着密切关系。自噬能协助机体清除病原体, 但有些细菌进化出多种策略干扰自噬信号通路或抑制自噬体与溶酶体融合形成自噬溶酶体来逃避自噬的降解, 甚至利用自噬来促进其生长增殖。文中从自噬的分子机制出发, 讨论多种致病菌与宿主细胞自噬关系的最新进展, 以及自噬与病原菌感染的作用和意义, 以期为病原菌感染导致的自噬研究提供参考。
关键词:自噬细菌感染ATG支原体
Fighting between bacteria and autophagy——death or rebirth Zhaodi Wang, and Honglei Ding
Zhaodi Wang, Honglei Ding
Laboratory of Veterinary Infectious Diseases, College of Animal Science and Technology, Southwest University, Chongqing
Received: December 8, 2019; Accepted: February 21, 2020; Published: April 28, 2020
Supported by: State Key Laboratory of Veterinary Biotechnology Foundation (No. SKLVBF201905), Fundamental Research Funds for the Central Universities (No. XDJK2020B012), Chongqing Technology Innovation and Application Development Project (No. cstc2019jscxmsxmX0402)
Corresponding author: Honglei Ding. Tel: +86-23-68251196; E-mail: hongleiding@swu.edu.cn.

Abstract: Autophagy is a highly conserved degradation process that targets cytoplasmic components, maintains metabolic stability in cells, and combates infection with various pathogenic bacteria.Autophagy can help body to eliminate invading pathogens; however, some bacteria have evolved multiple strategies to interfere with the autophagy signaling pathway or inhibit the fusion of autophagosomes with lysosomes to form autolysosomes to escape autophagic degradation, and even use autophagy to promote their growth and proliferation.This review discusses the newest progress in the relationship between pathogens and autophagy of host cell, and the role of autophagy in bacterial infection.We hope that this review provides useful knowledge for the research on autophagy caused by pathogenic infection.
Keywords: autophagybacteriainfectionATGMycoplasma
自噬(Autophagy)是细胞质中非特异性蛋白质被具有双层膜结构的自噬体(Autophagosome)包裹,与溶酶体融合形成自噬溶酶体(Autolysosome),最终将自噬溶酶体中包裹的物质降解的过程。1956年Clark用电镜观察新生小鼠肾组织时发现细胞中含有大量具有膜样结构的致密体,而且其中常含有类似于线粒体等的胞质结构[1]。在1963年的溶酶体国际会议上,Christian de Duve将细胞内包裹的胞质组分送入溶酶体的过程定义为自噬[2]。1984年,Rikihisa研究立克次体感染豚鼠多形核白细胞(Polymorphonuclear leukocytes,PMN)时发现溶酶体样物质,这种物质被称为自噬体,而未感染立克次体的PMN无自噬体[3]。这是关于细菌感染诱导细胞自噬的首次报道。此后,大量研究显示,细菌感染细胞后能导致细胞自噬的发生,进而抑制细菌的生长和增殖;反过来,一些细菌通过挟持自噬发生过程中的信号通路,抑制细胞吞噬或细胞凋亡等过程,避免细菌在胞内被宿主细胞杀灭,甚至利用自噬促进细菌在细胞内增殖[4]
基于自噬在病原菌与宿主细胞相互作用中扮演的重要角色,本文在简述自噬发生过程的基础上,综述了不同细菌在自噬过程中的分子和细胞事件(表 1),并对今后病原菌与宿主相互作用中自噬研究的发展方向和如何利用病原菌导致的自噬过程开发抗菌药物进行了展望。
表 1 病原菌与宿主自噬相互作用的机制Table 1 Mechanisms involved in the interaction of pathogenic bacteria with host autophagy
Pathogen G- or G+ Function (B: pathogenic bacteria; C: host cells) Reference
Staphylococcus aureus G+ C: Activation of MAPK14 and the phosphorylation of Atg5 inhibit the fusion of autophagic and lysosomes to escape the degradation of autophagy; PI3K/Akt pathway downregulates the autophagic level. [16, 19]
Group A Streptococcus G+ B: SpeB degrades SQSTM1, NDP52 and NBR1, preventing recognition by host cell autophagy-related proteins; SLO promote replication of GAS and help GAS escape to cytoplasm. C: Bcl-xL inhibites autophagy; Rab30 promote the removal of GAS in cell. [21-25]
Mycobacterium tuberculosis G+ B: Eis, Pkn G, Sap A and Hsp16.3 inhibite autophagy; CpsA promote MTB escape from LAP; MDP were recognized by Nod2 to enhance autophagy; some miRNA (such as miR-20a, miR-23a-5p, miR-30a, miR-33, miR-106b-5p, miR-125a-3p, miR-3619b-5p and miR-144) inhibite autophagy, miR-26a induces autophagy, miR-17-5p and miR-155 promote or inhibit autophagy in different conditions. C: IFN-γ is beneficial to clear MTB; 1, 25-D3 induce autophagy. [28-43]
Listeria monocytogenes G+ B: ActA, InlB, PLC and InlK help LM escape from autophagy, C: MAC-1 induce LAP to enhance the antibacterial ability. [44, 46-52]
Salmonella typhimurium G- B: SPI-1 and SPI-2. C: consumption of E3 ligases LRSAM1 and ARIH1 enhanced LUBAC-dependent ubiquitin, which inhibited the growth of Salmonella typhimurium in the cytoplasm. DAG is required for autophagy; galectin-8 restricts replication of Salmonella typhimurium in host cell. [54-61]
Shigella flexneri G- B: VirG binds to Atg5 to induce autophagy. C: inhibition of Rab1 activity blocks formation of autophagosomes. [63-64, 66-67]
Legionella pneumophila G- B:RavZ prevents formation of autophagosomes. Lpg1137 cuts Syntaxin 17 to block autophagy. [73-74]
Acinetobacter baumannii G- B: autophagy dependent on Sept 2 and Sept 9 C: Beclin-1/Atg7/Atg8、, MEK/ERK and AMPK-mTOR-ULK1 are involved in autophagy. [75]
Helicobacter pylori G- B: VacA inhibite autophagy. [77-78]
Mycoplasma gallisep G- C: ERK signaling pathway causes autophagy response. [79]
Mycoplasma ovipneumoniae G- C: NOD2 affects autophagy through JNK-Bcl-2/Beclinl pathway. [80-81]

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1 自噬发生的过程自噬过程包括一系列的步骤(图 1):①自噬的激活。不协调51样激酶1 (Uncoordinated-51-like kinase,ULK1)复合物是启动自噬的信号,它在募集自噬相关基因(Autophagy-related gene,Atg)蛋白到吞噬泡组装位点(Phagophore assembly site,PAS)过程中起关键作用。ULK1复合物包括ULK1、Atg13、Atg101和200 kDa的FAK家族激酶相互作用蛋白(FAK family kinase- interacting protein of 200 kDa,FIP200)[5]。在营养丰富的情况下,哺乳动物雷帕霉素靶蛋白复合物1 (Mammalian target of rapamycin complex 1,mTORC1)通过使ULK1和Atg13磷酸化,抑制ULK1复合物向PAS募集;相反,在营养缺乏或雷帕霉素处理情况下,mTORC1失活,ULK1被释放,从而将FIP200磷酸化并转移至PAS,并募集其他Atg蛋白为自噬体形成作准备[6]。②囊泡(Phagophore)成核。ULK1复合物将Beclin 1、Atg14样(Atg14-like,Atg14L)蛋白和磷酸肌醇3-激酶调节亚基4 (Phosphoinositide 3-kinase regulatory subunit 4,PIK3R4)募集到PAS,随后在此处启动囊泡成核过程。该过程在Ⅲ类PtdIns3激酶(PtdIns3-kinase classⅢ,PtdIns3KC3)的参与下在囊泡产生PtdIns3P,进而募集PtdIns3P结合蛋白,如WD-重复域磷酸肌醇相互作用1 (WD-repeat domain phosphoinositide-interacting 1,WIPI1)和WIPI2到PAS,并通过募集下游的Atg蛋白,如Atg16、Atg8、Atg9等,协助囊泡组装[7]。③自噬体的形成。自噬体的形成主要由两个泛素样连接系统所介导。第一个是Atg12-Atg5泛素样连接系统,它通过E1样酶Atg7和E2样酶Atg10激活,促进囊泡的延伸[8]。此外,Atg12-Atg5与Atg16L1形成的复合体与囊泡膜的扩展有关;当囊泡闭合形成自噬体时,该复合体从自噬体膜释放下来[9]。Atg16L1相关前体小泡之间的相互融合由可溶性N-乙基马来酰亚胺敏感因子黏附蛋白受体(Soluble N-ethylmaleimide-sensitive factor attachment protein receptors,SNARE)蛋白介导,该过程可能参与了囊泡的扩张过程。第二个是轻链3-磷脂酰乙醇胺(Light chain 3- Phosphatidylethanolamine,LC3-PE)泛素样连接系统。LC3和PE分别被E1样酶Atg7和E2样酶Atg3激活。Atg12-Atg5-Atg16L1复合物将LC3转移至囊泡膜,然后通过具有E3样酶活性的Atg12-Atg5与PE连接[10]。LC3-PE的形成还需要半胱氨酸蛋白酶Atg4B参与,因为Atg4B能够切割LC3的羧基末端,暴露甘氨酸残基后与PE共价结合[11]。此外,当自噬体完全形成后Atg4B解离一部分LC3-PE复合物,以便于LC3的循环,形成新的自噬体[12]。④自噬溶酶体的形成。自噬体通过与内体和溶酶体的一系列融合事件形成自噬溶酶体。在哺乳动物中,自噬体与溶酶体的融合需要小GTP酶Rab7、自噬体SNARE蛋白syntaxin 17、溶酶体SNARE囊泡相关膜蛋白8 (Vesicle-associated membrane protein 8,VAMP8)和一些溶酶体膜蛋白,如溶酶体相关膜蛋白2 (Lysosomal-associated membrane protein 2,LAMP2)[13-15]等的参与。⑤自噬溶酶体的降解。自噬溶酶体中的包裹物被降解成小分子物质后释放到细胞质中,这些小分子物质还可以被细胞重新利用。该降解过程依赖囊泡的酸化和多种溶酶体蛋白酶的参与。
图 1 细胞的自噬过程 Fig. 1 A diagram of the autophagy pathway.
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2 自噬与革兰氏阳性菌感染2.1 金黄色葡萄球菌金黄色葡萄球菌(Staphylococcus aureus,SA)感染可引起化脓性疾病和毒素性疾病。SA侵入HeLa细胞后被迅速泛素化,与具有结合泛素化底物的自噬受体螯合体1 (Sequestosome 1,SQSTM1)、核点蛋白52 (Nuclear dot protein 52,NDP52)和视神经蛋白(Optineurin,OPTN)结合诱导细胞自噬[16],其重要标志是SA与LC3形成明显的共定位,但是自噬体与溶酶体的融合受阻不能形成完整自噬流[17]。用SA感染牛乳腺上皮细胞(Bovine mammary epithelial cells,BMEC)后,BMEC中自噬标志蛋白LC3-Ⅱ的表达量升高,该研究在不同的细胞系中了证明了SA感染导致自噬[18]
SA可通过激活某些蛋白或信号通路逃避自噬。SA侵入HeLa细胞后,激活丝裂原活化蛋白激酶14 (Mitogen-activated protein kinase 14,MAPK14)、磷酸化Atg5来抑制自噬体与溶酶体的融合,逃避自噬的降解[16]。另外,SA感染小鼠巨噬细胞系RAW264.7导致的自噬与PI3K/Akt-Beclin1信号通路密切相关,抑制该信号通路后,SA诱导的自噬水平明显下调[19]。这些研究均显示,SA感染细胞后由于自噬体与溶酶体不能融合,形成不完整自噬,使自噬体不能被酸化,造成SA不被降解而在自噬体内增殖[17-19]
2.2 A群链球菌A群链球菌(Group A Streptococcus,GAS)是一种在自然界广泛存在的致病菌。GAS侵入宿主细胞后,胞质中大多数GAS包裹在含GAS的自噬体样空泡(GAS-containing autophagosome-like vacuoles,GcAVs)中,通过自噬途径降解;在无法形成自噬体的atg5-/-小鼠胚胎成纤维细胞(Mouse embryo fibroblasts,MEFs)中,GAS能够存活、增殖并从细胞中释放出来[20]。说明自噬对GAS的生长与增殖有抑制作用。
为了能够在细胞内长期生存,GAS演化出多种逃避自噬的策略。例如GAS利用某些毒力因子来逃避自噬的降解。链球菌致热外毒素B (Streptococcal pyrogenic exotoxin B,SpeB)是链球菌分泌的一种半胱氨酸蛋白酶,研究发现M1T1血清型GAS利用SpeB降解宿主细胞胞质中的SQSTM1、NDP52和BRCA 1相邻基因1 (Neighbor of BRCA 1 gene 1,NBR1)蛋白,阻止宿主细胞自噬相关蛋白的识别[21]。链球菌溶血素O (Streptolysin O,SLO)是胆固醇依赖的成孔细胞溶素保守家族的成员之一,GAS利用SLO在早期内体膜上形成孔隙,帮助细菌逃逸到细胞质中[22]。最近研究发现,SLO通过直接或间接促进β1整合素表达,募集NADPH氧化酶2(NADPH oxidase 2,NOX2)和产生活性氧(Reactive oxygen species,ROS),诱导产生LC3相关吞噬作用(LC3-associated phagocytosis,LAP),导致自噬溶酶体酸化不足,促进了GAS的增殖[23]
宿主细胞内的蛋白也对GAS诱导的自噬产生影响。抗凋亡Bcl-2家族成员Bcl-xL通过抑制自噬体-溶酶体融合直接抑制GAS诱导的自噬,并通过与Beclin 1-抗紫外线相关基因蛋白(Uv radiation resistance related gene protein,UVRAG)相互作用抑制GAS的内化作用,进而间接抑制GAS诱导的自噬[24]。GAS感染HeLa细胞后,Rab30通过募集β-磷脂酰肌醇4-激酶(Phosphatidylinositol 4-kinase beta,PI4KB),使之将磷脂酰肌醇(Phosphatidylinositol,PtdIns)转变为磷脂酰肌醇-4-磷酸(Phosphatidylinositol-4- phosphate,PtdIns4P),在PtdIns4P的参与下形成GcAVs,而GcAVs对于有效清除GAS具有重要作用[25]
2.3 结核分枝杆菌结核分枝杆菌(Mycobacterium tuberculosis,MTB)是一种胞内寄生菌。1971年,Armstrong等发现MTB能够抑制小鼠腹腔巨噬细胞中吞噬体与溶酶体融合[26]。巨噬细胞中MTB感染诱导的自噬可增强含分枝杆菌吞噬体的酸化作用和成熟,从而抑制胞内MTB的存活[27]。干扰素-γ (Interferon-γ,IFN-γ)在机体抵抗细菌感染过程中具有重要作用。在巨噬细胞中加入IFN-γ能促进LC3从胞质到内膜的转运,导致自噬发生;加入IFN-γ下游效应蛋白LRG-47,可发现自噬标记单丹磺酰戊二胺(Monodansylcadaverine)阳性小泡的形成[28]。进一步的研究发现,IFN-γ诱导产生鸟苷酸结合蛋白(Guanylate-binding proteins,Gbp),并利用Gbp具有促进宿主细胞对细菌的氧化杀灭作用和递呈抗菌肽至自噬溶酶体的功能,抑制胞内MTB的增殖,以保护宿主免受细菌感染[29],表明IFN-γ在MTB诱导自噬以及抑制胞内MTB的增殖中具有重要作用。
MTB能利用其分泌蛋白影响巨噬细胞的自噬发生和自噬体成熟,主要有4种分泌蛋白参与MTB诱导的巨噬细胞自噬:①增强型胞内生存蛋白(Enhanced intracellular survival protein,Eis)。该蛋白通过乙酰化激活c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)通路,降低ROS的水平,减弱自噬水平,增强胞内MTB的存活[30];②真核酶样丝氨酸/苏氨酸蛋白激酶G (Eukaryotic enzyme-like serine/threonine protein kinase G,Pkn G)。该蛋白通过破坏宿主细胞信号分子,抑制吞噬体-溶酶体融合,调节MTB的代谢和在胞内的存活[31-32];③分泌型酸性磷酸酶M (Secretory acid phosphatase M,SapM)。此蛋白通过水解磷脂酰肌醇3-磷酸(Phosphatidylinositol 3-phosphate,PI3P)抑制自噬体成熟,还通过与Rab7相互作用阻断自噬体与溶酶体融合抑制自噬流形成[33-35];④热休克蛋白16.3 (Heat shock protein 16.3,Hsp16.3)。该蛋白能干扰LC3表达以破坏自噬体的形成,从而抑制巨噬细胞对MTB的清除[36]
此外,MTB的细胞壁抗原以及DNA也能够影响自噬反应。研究发现,只有NADPH氧化酶通过其催化作用产生的ROS促进LC3-Ⅱ与吞噬体结合后,才能限制MTB的生存与增殖;MTB利用其胞壁蛋白CpsA阻断NADPH氧化酶的活化使其不能发挥作用,以逃避LAP的杀伤作用[37]。MTB感染巨噬细胞,核苷酸结合寡聚化结构域2(Nucleotide-binding oligomerization domain 2,Nod2)受体识别胞壁酰二肽(Muramyl dipeptide,MDP)产生大量IL-1β、IL-6和TNF-α等炎性因子,激活NF-κB通路,导致自噬标记蛋白LC3、Atg16L1和IRGM (Murine immunity related GTPases)的表达增加,诱导自噬[38-39]。巨噬细胞通过透化作用将MTB的DNA暴露,随后被干扰素基因刺激蛋白(Stimulator of interferon genes,STING)识别,在泛素连接酶作用和自噬蛋白(如NDP52和p62)介导下,含有MTB的吞噬体被泛素化,经泛素化的吞噬体与溶酶体融合形成吞噬溶酶体[40]
宿主细胞也能利用自身的维生素激活抗菌肽来影响自噬,进而影响MTB的生存与增殖。1, 25-二羟基维生素D3 (1, 25-dihydroxyvitamin D3,1, 25-D3)是维生素D3的活性形式,Yuk等研究发现,1, 25-D3通过对MTB有杀伤作用的抗菌蛋白cathelicidin诱导人单核细胞自噬,从而激活自噬相关基因beclin 1atg5的转录;1, 25-D3还依赖cathelicidin诱导分枝杆菌吞噬体与自噬体的共定位[41]。在细胞内MTB诱导LAP的过程中,Ⅲ类PI3K复合物的募集增加了PI3P的产生,使NOX2复合物稳定产生ROS并募集自噬蛋白Atg5、Atg12、Atg16L、Atg7和Atg3,还将脂化的LC3-Ⅱ与吞噬体膜结合,促进细胞自噬[42]
微小RNA (MicroRNA,miRNA)是一种由21–-23个核苷酸组成的单链非编码RNA,通过形成RNA沉默复合体等方式与一些mRNA的3'非编码区(3' untranslated region,3'UTR)结合,阻止mRNA翻译并将其降解,调控转录后的蛋白表达。巨噬细胞内的miRNA在MTB诱导细胞自噬过程中起重要作用。大部分miRNA,如miR-20a、miR-23a-5p、miR-30a、miR-33、miR-106b-5p、miR-125a-3p、miR-3619b-5p和miR-144等,具有抑制细胞自噬、增强MTB在细胞内存活的能力;有的miRNA,如miR-26a,则促进细胞自噬,抑制胞内MTB的生长;个别miRNA,如miR-17-5p和miR-155,通过靶向调控/负调控不同的自噬相关基因,促进或抑制自噬过程,进而抑制或促进MTB在细胞内的存活[43]
2.4 产单核细胞李斯特菌产单核细胞李斯特菌(Listeria monocytogenes,LM)是一种可胞内寄生的人兽共患病病原菌。宿主细胞感染LM后,通过自身的识别受体激活自噬。β2整合素巨噬细胞抗原1 (Macrophage antigen 1,MAC-1)是巨噬细胞表达的一种识别多种微生物配体的受体。研究表明,宿主细胞的MAC-1通过激活酸性鞘磷脂酶(Acid sphingomyelinase,ASM)和烟酰胺腺嘌呤二核苷酸磷酸2 (Nicotinamide adenine dinucleotide phosphate 2,NADH2)产生ROS,诱导LAP。而LAP促进含LM的吞噬体与溶酶体的融合,从而增强巨噬细胞的抗菌能力和机体的免疫应答[44]。TLR2参与LM产生的自噬反应,在tlr2-/-巨噬细胞中,LM与LC3不能形成共定位;进一步研究证明TLR2通过其下游的ERK信号通路参与这一自噬过程[45]
LM侵入宿主细胞后,类似于GAS,也能够利用其毒力因子协助其从自噬体中逃逸,然后进入胞质中继续增殖。其毒力因子李斯特菌溶血素O (Listeriolysin O,LLO)是一种胆固醇依赖的成孔细胞溶素。LM进入巨噬细胞后,在感染早期,依赖于LLO诱导自噬,LM的增殖受到抑制[46];感染一段时间后,在两种磷脂酶C (Phospholipase C,PLC),——磷脂酰肌醇磷脂酶C (Phosphatidylinositol phospholipase C,PI -PLC)和磷脂酰胆碱特异磷脂酶C (Phosphatidylcholine- specific phospholipase C,PCPLC)的帮助下,逃逸出自噬体[46-47]。LLO则与胆固醇结合插入宿主细胞膜,聚合形成孔隙,以利于LM从细胞中逃逸[48-49]。因此,依赖于LLO诱导的自噬反应在LM的感染中随着时间延长呈现动态变化。肌动蛋白A (Actin A,ActA)是LM的一种重要的毒力因子,参与了LM逃避宿主细胞的自噬。敲除actA基因能够降低LM感染细胞后自噬的发生[46];该过程是ActA通过将肌动蛋白相关蛋白2/3 (Actin-related proteins,Arp2/3)复合物和可提取核抗原/血管舒张剂刺激磷蛋白(Extractable nuclear antigen/vasodilator-stimulated phosphoprotein,Ena/VASP)募集到细菌表面,这两种蛋白伪装成细菌,从而使细菌不被自噬识别[50]。LM还利用其表面的内化素K (Internalins K,InlK)将穹隆蛋白(Major vault protein,MVP)招募到细菌表面,随后通过ActA取代Inlk、肌动蛋白取代MVP来修饰细菌,阻止泛素蛋白的招募、p62的识别和LC3的募集,帮助LM逃避自噬[51-52]。除了ActA蛋白,在感染后期,PI -PLC和PC –PLC在自噬逃逸中也发挥重要作用[39]
3 自噬与革兰氏阴性菌感染3.1 沙门氏菌沙门氏菌(Salmonella)主要引起人和哺乳动物的胃肠炎。沙门氏菌包含两种Ⅲ型分泌系统(Type Ⅲ secretion system,T3SS),分别为沙门氏菌毒力岛1 (Salmonella pathogenicity island 1,SPI-1)和SPI-2。SPI-1对于沙门氏菌的侵袭是必不可少的,SPI-2则促进沙门氏菌在胞内的存活。
自噬通过两种方式作用于沙门氏菌。沙门氏菌寄居的吞噬泡称为含沙门氏菌液泡(Salmonella- containing vacuole,SCV)。大部分沙门氏菌存在于SCV中,只有一小部分从SCV逃逸到细胞质中复制。从SCV逃逸的沙门氏菌被泛素化,泛素化修饰招募多种自噬衔接蛋白,包括SQSTM1/p62、NDP52和OPTN等,这些蛋白通过泛素化结合结构域与发生泛素化的细菌结合,然后再通过与自噬体膜锚定的Atg8家族成员相互作用连接新生的自噬体。含沙门氏菌的自噬体与溶酶体融合后在水解环境下被杀死。另一种方式是SPI-1在SCV打孔,造成受损的SCV被自噬识别。在这种情况下,沙门氏菌不需要进入细胞质被自噬捕获。SPI-1在SCV形成小孔导致多种胞质半乳糖凝集素(Galectin)的聚集,如监测细胞内溶酶体完整性(Endolysosomal integrity)的galectin-8。SCV经galectin-8修饰后招募泛素连接酶和NDP-52到细胞膜,从而使SCV成分被自噬清除[53]
自噬识别细菌的过程通常与泛素化作用相关,而E3连接酶对细胞自噬过程中的泛素化作用具有重要意义。其中人亮氨酸富集不育α模体1 (Human leucine rich repeat and sterile α motif containing 1,LRSAM1)通过其富含亮氨酸的重复序列(Leucine rich repeat,LRR)结构域和RING (Really interesting new gene)结构域识别HeLa细胞胞质中鼠伤寒沙门氏菌,启动泛素依赖的自噬反应,从而抑制鼠伤寒沙门氏菌感染[54]。另外E3连接酶线性泛素链组装复合物(Linear ubiquitin chain assembly complex,LUBAC)在鼠伤寒沙门氏菌的表面产生线性多聚泛素,募集受体OPTN、NDP52和SQSTM1/p62,从而限制鼠伤寒沙门氏菌的增殖[55]。还有RBR (Ring- between-Ring) E3连接酶ARIH1可直接将鼠伤寒沙门氏菌靶向自噬体[56]。鼠伤寒沙门氏菌感染细胞后,LRSAM1和ARIH1的消耗致使LUBAC依赖的泛素化增强,从而抑制细胞质中鼠伤寒沙门氏菌的生长[55-57]。说明宿主细胞能够通过在细菌表面募集不同的泛素连接酶启动自噬,以阻止细胞内鼠伤寒沙门氏菌的增殖。
除了泛素化作用,脂类在鼠伤寒沙门氏菌感染诱导的自噬过程中也发挥重要作用。鼠伤寒沙门氏菌感染诱导的自噬中需要甘油二酯(Diacylglycerol,DAG)参与,而DAG则需要磷脂酶D (Phospholipase D,PLD)和磷脂酸磷酸酶(Phosphatidic acid phosphatase,PAP)的参与产生[58]。半乳糖凝集素(Galectin)是一种β-半乳糖苷结合凝集素,存在于胞质中。galectin-8能够通过与暴露在受损的SCV表面的宿主聚糖结合来监测内体/溶酶体的完整性和监测鼠伤寒沙门氏菌的入侵,进而galectin-8与NDP52结合形成异源二聚体并导致NDP52发生泛素化后与自噬标志蛋白LAMP1结合,从而导致自噬发生,限制鼠伤寒沙门氏菌在胞内的增殖[59]
鼠伤寒沙门氏菌感染细胞后通过激活胞内一些信号通路来影响自噬反应。鼠伤寒沙门氏菌感染细胞后导致短暂的胞内能量缺失,激活AMP依赖的蛋白激酶(Adenosine 5'-monophosphate (AMP)-activated protein kinase,AMPK),启动自噬发生。这一过程是通过胞内肝激酶B1(Liver kinase B1,LKB1)复合体(包括Sirt1和LKB1)对AMPK活性的调控来实现的。鼠伤寒沙门氏菌进入细胞后,某些毒力因子和Sirt1相互作用,使Sirt1将LKB1的乙酰基脱去,去乙酰化的LKB1活化后进一步激活AMPK[60]。也有研究发现,鼠伤寒沙门氏菌感染RAW 264.7细胞能激活多种Toll样受体(Toll-like receptor,TLR),TLRs激活TGF-β-活化激酶1 (TGF-β-activated kinase 1,TAK1)后将AMPK活化,从而诱导细胞自噬;抑制TAK1活性后则阻断了细胞自噬,有利于细胞内鼠伤寒沙门氏菌的生长。这些结果表明,鼠伤寒沙门氏菌通过TLRs/TAK1/AMPK信号通路导致自噬发生,清除胞内鼠伤寒沙门氏菌[61]。尽管细胞内Sirt1和TLRs都可以诱导AMPK通路的细胞自噬,但对细胞内鼠伤寒沙门氏菌存活的影响迥异,导致这两种不同结果的原因有待进一步研究。
3.2 福氏志贺氏菌福氏志贺氏菌(Shigella flexneri)是一种肠道致病菌,可引起人的痢疾。Nod1和Nod2可募集Atg16L至细胞表面,然后福氏志贺氏菌在Atg16L定位区域进入细胞后引起自噬[38]。在此过程中,在线粒体的协助下,细胞骨架蛋白Septins形成牢笼样结构将福氏志贺氏菌限制在其内部,并抑制了福氏志贺氏菌的增殖。而当线粒体破裂之后,福氏志贺氏菌则能从Septin牢笼样结构中逃逸出来[62]。福氏志贺氏菌可通过其VirG蛋白与Atg5蛋白结合来诱导自噬发生[63]。膜融合相关蛋白Tecpr1 (Tectonin domain-containing protein)和Atg5蛋白均位于吞噬泡上,它与WIPI-2相互作用促进含志贺氏菌囊泡成核和自噬发生,该过程是通过Tecpr1-Atg5与VirG蛋白作用实现的[64]。福氏志贺氏菌T3SS分泌的IcsB蛋白也能与Atg5结合,且其与Atg5的结合位点和VirG与Atg5的结合位点相同。因此,福氏志贺氏菌可通过分泌IcsB并与VirG竞争性结合Atg5阻断自噬发生,使得福氏志贺氏菌能逃避自噬降解[63],IcsB的胆固醇结合结构域(Cholesterol binding domain,CBD)在其介导的自噬逃逸中发挥重要作用[65]。Rab1是细胞内一种小Rab GTP酶,不但介导蛋白质从内质网到高尔基体运输,最近发现其对自噬体形成也是必不可少的[66-67]。抑制Rab1的活性,能够阻断福氏志贺氏菌感染细胞中自噬体形成[68]
3.3 嗜肺军团菌嗜肺军团菌(Legionella pneumophila)是一种革兰氏阴性致病菌,可引起人致死性肺炎或流感样症状。嗜肺军团菌进入宿主细胞吞噬体后,Atg7蛋白马上与之结合,然后与Atg8结合形成含军团菌液泡(Legionella-containing vacuole,LCV)[69-70]。LCV可与内质网来源的囊泡融合,有利于嗜肺军团菌在其中的复制,融合过程需要Rab1和Sec22b蛋白的参与[71]
SNARE蛋白Syntaxin 17在自噬体膜形成过程中发挥重要作用,它可以将Atg14L蛋白募集到内质网-线粒体结合位点,以帮助其形成自噬体[72]。另外Syntaxin 17对于自噬体和溶酶体的融合也是必不可少的,其功能发挥依赖于其C端的发夹结构[14]。嗜肺军团菌的效应蛋白Lpg1137是一种丝氨酸蛋白酶,它可切割Syntaxin 17,进而阻断自噬发生[73]。此外,RavZ蛋白能够阻断自噬体膜上的Atg8 (LC3)蛋白PE的结合,其过程是RavZ水解Atg8 C端的甘氨酸残基和相邻的芳香族残基之间的酰胺键,使之不能脂化和PE形成LC3-PE复合体在囊泡聚集,进而阻断自噬体形成[74]
3.4 鲍曼不动杆菌鲍曼不动杆菌(Acinetobacter baumannii,AB)是一种机会致病菌。AB诱导的自噬是通过泛素E3连接酶LRSAM1与泛素化蛋白包绕菌体,并与募集的自噬配体蛋白NDP52及SQSTM1/p62作用而启动的。敲除骨架蛋白基因sept 2sept 9后,SQSTM1和NDP52则不能在菌体周围募集,细胞自噬受到抑制,说明AB诱导的自噬依赖于骨架蛋白成分。Beclin-1/Atg7/Atg8、MEK/ERK及AMPK/mTOR/ULK1信号通路参与了AB诱导的自噬反应的发生[75]
3.5 其他革兰氏阴性菌牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)感染宿主细胞诱导自噬,但自噬途径取决于宿主细胞类型。例如在胎牛心脏内皮细胞(Fetal bovine heart endothelial cells,FBHEC)和牛主动脉内皮细胞(Bovine aortic endothelial cells,BAEC)诱导的自噬中Pg存在于多膜囊泡,而人冠状动脉内皮细胞(Human coronary artery endothelial cells,HCAEC)中则存在于自噬体[76]。Pg感染后能够阻断自噬体与溶酶体融合[76]
幽门螺杆菌(Helicobacter pylori,Hp)感染人单核细胞THP-1能诱导自噬体形成,并在其中增殖;vacAcagA缺失菌株中,Hp的增殖能力下降[77]。进一步研究发现,VacA通过改变细胞内氨基酸平衡抑制mTORC1的活性,使mTORC1从溶酶体表面解离,导致ULK1复合物活化,引起自噬[78]。CagA在自噬中的作用还未见报道。
4 自噬与支原体感染与其他病原菌相比,关于自噬与支原体感染之间关系的研究还较少。鸡毒支原体(Mycoplasma gallisepticum,MG)感染RAW264.7后可诱导自噬,该自噬反应经ERK信号通路激活,TLR2则调节MG感染经ERK信号通路引起的自噬反应[79]。绵羊支原体(Mycoplasma ovipneumoniae,MO)感染小鼠上皮细胞TC-1诱导的自噬参与宿主细胞对MO的清除,该自噬反应经JNK-Bcl-2/Beclin l信号通路完成,且受Nod2调控[80-81]。Raymond等人研究证明,猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)能够进入细胞内[82]。本实验室验证了Raymond等人的研究结果,发现在Mhp感染猪肺组织的临床样本中,与Mhp阴性样品相比,LC3-Ⅱ的表达量明显升高,且与Mhp形成共定位;Mhp感染猪肾传代细胞PK-15能显著增加自噬标志蛋白的表达,说明Mhp感染宿主细胞也能导致自噬发生。此外还发现自噬体与溶酶体不能融合形成完整的自噬流,这造成了Mhp在细胞内随着感染时间延长大量增殖,表明自噬促进了Mhp在细胞内的增殖,但这种自噬反应是通过哪条信号通路介导还需进一步明确。自噬体与溶酶体的融合需要SNARE复合物(包括syntaxin 17、SNAP29、VAMP8等蛋白)和Rab7、LAMP2的参与,SNAP47、ATG14也参与了这一融合过程。Mhp如何与这些蛋白相互作用阻断自噬体与溶酶体的融合也是本实验室下一步要开展的工作。
5 总结与展望细菌感染宿主细胞后,自噬和细菌相互作用的结果有:①阻止细菌增殖,或是自噬通过与其他免疫因子(如炎症因子、IFN-γ等)相互作用,以控制细菌的感染;②细菌利用其毒力因子、合成miRNA或分泌某些蛋白质和脂质等来阻断完整的自噬过程,从而逃避自噬对其增殖或杀灭作用。虽然已经有很多细菌导致自噬的报道,但大部分研究还仅仅是自噬现象的发现。细菌进入细胞引起的自噬是导致细菌被杀灭,还是促进了细菌的生长和繁殖,或者二者兼具;造成细菌杀灭或增殖的机制是什么?这是研究细菌与自噬关系需要进一步开展的工作。自噬现象往往不是独立存在的,其与泛素化、炎症、免疫反应、miRNA等存在千丝万缕的联系,深入探讨自噬与其相互关系是了解细菌致病机制的重要窗口。细菌中的哪些成分在其引起的自噬中发挥重要作用也是细菌引起自噬研究的短板之一。构建基因突变菌株或基因转染等是研究细菌蛋白在自噬中发挥功能的重要手段。明确细菌感染导致自噬的分子过程和在致病中发挥重要功能的细菌蛋白在自噬过程中的作用对预防性生物制品、小分子靶向抗菌药物的开发具有重要的理论指导意义。
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