【作者】 吕杰；

【导师】 黄瑞；

【作者基本信息】 苏州大学 ， 病原生物学， 2012， 博士

【摘要】 目的：通过建立沙门菌感染的细胞模型,观察伤寒沙门菌质粒pRST98及与其密切相关的沙门菌质粒毒力基因(Salmonella plasmid virulence gene B, spvB)对自噬及感染结局的影响,探讨其发生机制及相关信号通路,以期揭示沙门菌质粒毒力基因增强宿主菌毒力的分子机制,为探索通过调控细胞自噬水平控制沙门菌感染的新途径,并为后续新药靶点的发现及疫苗开发提供实验和理论依据。方法：1.沙门菌质粒对细胞自噬及感染结局的影响。以携带质粒PRST98的伤寒沙门菌(Salmonella typhi, S.typhi)野生株ST8为实验株,消除pRST98的突变株PRST98为阴性对照株,携带沙门菌质粒毒力基因(Salmonella plasmid virulence gene, spv)的鼠伤寒沙门菌标准毒株(Salmonella typhimurium)SR-11为阳性对照株,将受试菌与野生型小鼠胚胎成纤维细胞(wide type mouse embryonic fibroblasts, WT-MEFs)及自噬基因5缺陷型小鼠胚胎成纤维细胞(autophagy associated gene5deficient mouse embryonic fibroblasts, Atg5-/-MEFs)体外共培养,制作细胞感染模型。以对数生长期细菌按感染复数(multiplicity of infection, MOI)为100：1感染细胞,同时设立WT-MEFs的自噬激动剂——雷帕霉素(rapamycin, RAPA)干预组。将细胞与细菌共作用1h后吸取上清,此时定为“0"点,加入含有100μg/ml的阿米卡星(Amikacin, AMK)培养液作用2h以去除胞外菌,然后将AMK浓度降为10μg/ml以抑制从感染细胞中释放至培养液中的细菌生长。分别在感染的不同时间段(1h、3h、5h、8h和10h)收获细胞,采用以下方法进行实验检测：①单丹磺酰尸胺(monodansylcadaverin, MDC)染色,荧光显微镜下观察细胞内点状自噬囊泡；②透射电镜(transmission electron microscope, TEM)观察细胞内双层或多层膜结构的自噬小体；③Western Blotting (WB)检测细胞自噬标志蛋白——微管相关蛋白1轻链3-Ⅱ(Microtubule-associated protein1light chain3-Ⅱ, MAP1-LC3-Ⅱ,即LC3-Ⅱ)表达情况；④Annexin.V-FITC/Propidium Iodide (Ann.V/PI)双染流式细胞术(flow cytometry, FCM)分析细胞凋亡率；⑤梯度稀释平板菌落计数法检测胞内集落形成单位(colony forming unit, CFU)。2.沙门菌质粒毒力基因spvB影响细胞自噬及机制研究。以spvB缺陷的鼠伤寒沙门菌SR-11突变株(SR-11-△spvB)及含有spvB的野生型鼠伤寒沙门菌标准毒株SR-11(SR-11)为研究对象,将实验菌株体外感染WT-MEFs及Atg5-/-MEFs, MOI为100：1,细胞感染过程同第一部分。分别在感染的不同时间段(1h、3h、5h、8h和10h)收获细胞,进行以下实验：①WB检测细胞LC3-Ⅱ蛋白、磷脂酶D(phospholipase D1——PLD1和phospholipase D2——PLD2)蛋白及Toll样受体4(Toll-like receptors4, TLR4)蛋白表达情况；②放射性[9,103H]油酸标记检测感染细胞的PLD活性；③逆转录聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)检测细胞TLR4信使RNA (messanger RNA, mRNA)表达情况；④Ann.V/PI双染FCM分析细胞凋亡率；⑤酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测细胞Thl型细胞因子干扰素-γ(Interferon-γ, IFN-γ)及Th2型细胞因子白细胞介素-4(interleukin-4, IL-4)和白细胞介素-13(interleukin-13, IL-13)的表达；⑥梯度稀释平板菌落计数法检测胞内CFU。结果：1.沙门菌质粒对细胞自噬及感染结局的影响(1) MDC荧光染色结果：ST8及SR-11感染的各组细胞中,在感染早期,WT-MEFs内可见很少量的点状自噬囊泡颗粒,RAPA干预后,WT-MEFs在感染早期1h点状自噬囊泡颗粒数量增多。感染早期1h,ST8-△pRST98感染的WT-MEFs出现大量的点状自噬囊泡颗粒,3h时颗粒数量减少,RAPA干预的ST8-ApRsT98感染WT-MEFs在早期亦出现数量众多的自噬囊泡颗粒。(2)细胞超微结构观察：在感染早期1h, ST8及SR-11感染的WT-MEFs内未见自噬小体,RAPA干预后,ST8及SR-11感染的WT-MEFs胞内可见双层膜结构的自噬小体。感染早期1h,电镜下可见ST8-△pRST98感染的WT-MEFs及RAPA干预的WT-MEFs内有典型的双层或多层膜自噬小体。(3) LC3-Ⅱ蛋白表达：在感染早期1h,ST8及SR-11感染WT-MEFs的LC3-Ⅱ表达较弱,RAPA干预后,ST8及SR-11感染WT-MEFs的LC3-Ⅱ表达明显增强。感染中晚期5h-10h, LC3-Ⅱ在上述各组感染细胞均不表达。感染早期1h和3h,ST8-△pRST98感染的WT-MEFs的LC3-Ⅱ表达较强,且LC3-Ⅱ在1h的表达强度高于3h; RAPA干预WT-MEFs后,LC3-Ⅱ表达明显增加。(4)3株细菌感染的Atg5-/-MEFs在感染各时间段均没有观察到点状自噬囊泡颗粒、双层膜结构的自噬小体及LC3-Ⅱ表达等自噬现象发生。(5)细胞凋亡检测结果：ST8感染的各组细胞中,Atg5-/-MEFs和WT-MEFs之间凋亡率无显著性差异,RAPA干预WT-MEFs后,其凋亡率明显降低。SR-11感染各组细胞的凋亡比较结果与ST8感染细胞类似,Atg5-/-MEFs和WT-MEFs之间凋亡率无显著性差异,在以RAPA干预后,WT-MEFs凋亡率显著降低。ST8-△pRST98感染的各组细胞凋亡结果分析显示,在各个时间段,Atg5-/-MEFs凋亡率最高,RAPA干预的WT-MEFs凋亡率高于RAPA未干预的WT-MEFs凋亡率。(6)梯度稀释法计数胞内细菌数量：ST8感染的各组细胞之间,Atg5-/-MEFs和WT-MEFs的胞内细菌数量无显著性差异,而RAPA干预的WT-MEFs胞内细菌数量显著低于RAPA未干预WT-MEFs组。SR-11感染各组细胞的胞内细菌数量比较结果与ST8感染细胞类似,Atg5-/-MEFs和WT-MEFs之间无显著性差异,RAPA干预后,WT-MEFs内细菌数量明显减少。ST8-△pRsT98感染的各组细胞比较结果显示,Atg5-/-MEFs胞内细菌数量最多,RAPA干预的WT-MEFs与RAPA未干预的WT-MEFs之间无显著性差异。2.沙门菌质粒毒力基因spvB影响细胞自噬及机制研究(1)感染细胞LC3-Ⅱ蛋白表达情况：在感染早期1h和3h, SR-11感染WT-MEFs的LC3-Ⅱ表达明显弱于SR-11-△spvB感染的WT-MEFs,在感染中后期5h～10h,LC3-Ⅱ均不表达。(2)感染细胞PLD1和PLD2蛋白表达情况：在各时间段,SR-11感染细胞的PLD1蛋白表达水平强于SR-11-△spvB感染细胞,而PLD2蛋白表达水平在两株细菌感染细胞之间无显著性差异。(3)感染细胞TLR4蛋白表达情况：在感染各时间段,SR-11感染细胞的TLR4蛋白表达水平明显弱于SR-11-△spvB感染细胞。(4)感染细胞PLD活性测定：SR-11及SR-11-△spvB感染细胞的PLD活性均明显高于空白细胞的PLD基础活性；在感染各时间段,SR-11感染细胞的PLD活性均高于SR-11-△spvB感染细胞,1h达到高峰,以后逐渐下降。(5)感染细胞TLR4mRNA表达情况：在感染各时间段,SR-11感染细胞的TLR4mRNA表达水平低于SR-11-△spvB感染细胞。(6)感染细胞凋亡率检测：在感染各时间段,SR-11-△spvB感染的WT-MEFs凋亡率显著低于SR-11感染的WT-MEFs及SR-11-△spvB感染的Atg5-/-MEFs凋亡率；SR-11感染的WT-MEFs与Atg5-/-MEFs之间,凋亡率无显著性差异。(7)感染细胞的细胞因子表达情况：感染早期和中期(1h～5h),SR-11感染细胞的IFN-γ表达水平低于SR-11-△spvB感染细胞组,而SR-11感染细胞的IL-4及IL-13表达明显高于SR-11-△spvB感染细胞组；在感染后期(8h～10h),两株细菌感染细胞的细胞因子表达无显著性差异。(8)胞内细菌数检测：SR-11感染的WT-MEFs及Atg5-/-MEFs之间胞内细菌数量无显著性差异。SR-11-△spvB感染WT-MEFs的胞内细菌数量显著低于SR-11-△spvB感染的Atg5-/-MEFs和SR-11感染的WT-MEFs胞内细菌数。结论：1.伤寒沙门菌质粒pRST98可通过抑制细胞自噬促进细菌在宿主细胞内的存活和增殖,从而加重细胞损伤。2.伤寒沙门菌质粒pRST98抑制自噬的作用与其基因序列上的重要毒力基因spvB密切相关。spvB可通过抑制细胞TLR4表达,促进细胞PLD1表达及增加PLD活性,抑制Th1型细胞因子IFN-γ、促进Th2型细胞因子IL-4和IL-13表达从而导致Th1/Th2免疫漂移等途径抑制细胞自噬。spvB抑制自噬的信号通路与丝氨酸／苏氨酸激酶(serine/threonine protein kinase, AKT)依赖性方式激活TOR激酶(Mammalian Target of Rapamycin, mTOR)密切相关。3.作用于mTOR靶点的药物——雷帕霉素可减弱沙门菌质粒毒力基因对自噬的抑制作用,从而增强宿主细胞对感染病原菌的杀伤能力,减轻细胞损伤。4.自噬在沙门菌感染中具有“双刃剑”的作用,适度激活有利于清除胞内感染病原菌,减轻细胞损伤；而其缺失、抑制或过度增强可加重细胞损伤和感染。更多还原

【Abstract】 Objective:To observe the effect of Salmonella virulence plasmid pRST98and Salmonella plasmid virulence gene B (spvB) on cellular autophagy and infection outcome through cells model infected by Salmonella infection in vitro, therefore discuss the molecular mechanism and possible signaling pathway. This study can help to elucidate the virulent mechanism of pRST98in Salmonella infection; also it can provide the experimental and theoretical basis on regulating autophagic pathway, discovering new drug target sites and vaccine exploration for controlling Salmonella infection.Methods:1. The study of cellular autophagy and infection outcome influenced by Salmonella virulence plasmid. In this study, Salmonella typhi (S. typhi) strain ST8carrying a virulence plasmid pRsT98was used as an experimental strain, pRST98-deletion S. typhi strain ST8-△pRST98was used as a negative control and standard virulent strain Salmonella typhimurium (S. typhimurium) SR-11containing Salmonella plasmid virulence gene (spv) presented on a100Kb virulent plasmid was used as a positive control. Three kinds of bacteria were added to wide type mouse embryonic fibroblasts (WT-MEFs) and autophagy associated gene5deficient mouse embryonic fibroblasts (Atg5-/-MEFs) in vitro respectively at a multiplicity of infection (MOI) of100:1, meanwhile the groups of infected-WT-MEFs treated by autophagy inducer rapamycin (RAPA) were set up. After incubated at37℃for1h (0-h time point), infected cells were washed three times with sterile phosphate buffered saline (PBS); then DMEM completely containing amikacin (100μg/ml) was added to kill remaining extracellular bacteria. After2h of further incubation at37℃, medium in the plates was replaced with DMEM containing amikacin (10μg/ml) to inhibit the propagation of possible extracellular bacteria in the medium. Infected cells were collected and detected at1h,3h,5h,8h and10h time points of infection. Autophagic vacuoles of infected cells were visualized under fluorescence microscopy by monodansycadaverine (MDC) staining. The gold standard of autophagy phenomenon such as double-or multilayer membrane of autophagosome in the cytoplasma was observed by transmission electron microscopy (TEM). The expression of autophagic protein LC3-II was detected by Western Blotting (WB) assay. The apoptotic rate of infected cells was analysized by flow cytometry (FCM) through the method of Annexin.V-FITC (Ann. V)/Propidium Iodide (PI) double staining. The number of bacteria inside infected cells was quantitied by plate count method.2. The effect of spvB on cellular autophagy and its molecular mechanism discussion. WT-MEFs and Atg5-/-MEFs were co-cultured with spvB mutant of SR-11strain (SR-11-AspvB) and wide type of SR-11strain containing spvB (SR-11) in vitro. Two kinds of bacteria were added to WT-MEFs and Atg5-/-MEFs at MOI of100:1. After incubated at37℃for1h (0-h time point), infected cells were washed three times with PBS; then DMEM completely containing amikacin (100μg/ml) was added to kill remaining extracellular bacteria. After2h of further incubation at37℃, medium in the plates was replaced with DMEM containing amikacin (10μg/ml) to inhibit the propagation of possible extracellular bacteria in the medium. Infected cells were collected at1h,3h,5h,8h and10h and detected by the following methods. The expression of LC3-Ⅱ protein, phospholipase D (PLD1and PLD2) protein and Toll-ike receptor4(TLR4) protein of infected cells were identified with WB assay. PLD activity of infected cells was detected by radioactive [9,10-3H] oleic acid labeling. The expression of TLR4mRNA in infected cells was semiquantitied by reverse transcription polymerase chain reaction (RT-PCR) assay. Apoptosis of infected cells was detected by FCM through Ann. V/PI double labeling. Thl-type cytokine secretion such as interferon-γ (IFN-γ) of infected cells and Th2-type cytokines such as interleukin-4(IL-4) as well as interleukin-13(IL-13) of infected cells were examined by enzyme-linked immunosorbent assay (ELISA). The number of bacteria inside infected cells was quantitied by plate count method.Results:1. The effect of Salmonella virulence plasmid on cellular autophagy and infection outcome.(1) Results of MDC fluorescent staining under fluorescene microscopy. Only few sporadic autophagic vesicles were observed in the cytoplasm of WT-MEFs infected by and SR-11at1h, while autophagy inducer RAPA-treatment induced lots of autophagic vesicles to appear in WT-MEFs which were infected by ST8and SR-11at1h. In the cytoplasm of WT-MEFs and RAPA-treated WT-MEFs infected by ST8-△pRST98at1h, a few typical autophagic vesicles could be observed, while the number of autophagic vesicles at3h was fewer than at1h.(2) Ultrastructure of infected cells under TEM. WT-MEFs infected by ST8and SR-11did not appear typical autophagic vacuoles in the cytoplasm, however, ST8and SR-11infected-WT-MEFs treated by RAPA displayed the trypical autophagic vacuoles with double membrane at1h. WT-MEFs and RAPA-treated WT-MEFs infected with ST8-△pRST98showed typical autophagic vacuole with double-or multilayer membrane in the cytoplasm at1h.(3) Expression of autophagic protein LC3-Ⅱ in infected cells. LC3-Ⅱ protein expression in WT-MEFs infected by ST8containing pRST98and SR-11containing spv was weaker than in WT-MEFs infected by ST8-△pRST98at1h; however, this infection-mediated suppression was reversed with RAPA-treatment as shown by the LC3-II protein expression in both RAPA-treated WT-MEFs infected by ST8and SR-11. WT-MEFs infected by ST8-△pRST98showed stronger LC3-Ⅱ protein expression at1h and3h than WT-MEFs infected by ST8and SR-11, and the intensity of LC3-Ⅱ protein expression at1h was stronger than at3h. LC3-Ⅱ protein expression in RAPA-treatment WT-MEFs infected by ST8-△pRST98was higher than in RAPA-untreated WT-MEFs. The intensity of LC3-II protein expression in all infected cells at3h was weaker than at1h, and disappeared from5h to10h.(4) Punctate autophagic vesicles, LC3-Ⅱ protein expression and typical autophagic vacuole with double-or multilayer membrane could not been observed in Atg5-/-MEFs infected by three kinds of bacteria at each time points of infection.(5) Apoptotic rate of infected cells. Among cells infected by ST8containing pRsT98, the apoptotic rate in WT-MEFs had no significant difference than in Atg5-/-MEFs; while the apoptotic rate in RAPA-treatment WT-MEFs was significant lower than in RAPA untreated-WT-MEFs. The results of apoptotic rate among cells infectd by SR-11contaning spv showed similar as caused by ST8. The apoptotic rate in WT-MEFs had no significant difference than in Atg5-/-MEFs infected by SR-11; while RAPA treatment significant decreased apoptosis in WT-MEFs than in RAPA-untreated WT-MEFs infected by SR-11. The apoptotic rate in Atg5-/-MEFs was highest among cells infected by ST8-△pRST98, and the apoptotic rate in RAPA untreated-WT-MEFs was lower than in RAPA-treatment WT-MEFs infected by ST8-ApRST98.(6) The number of bacteria inside infected cells. Among cells infected by ST8, the number of intracellular bacteria in WT-MEFs was similar to the bacterial quantity in Atg5-/-MEFs, while WT-MEFs with RAPA-treatment had less intracellular bacterial quantity than RAPA-untreated WT-MEFs. Among cells infected by SR-11containing spv, the number of intracellular bacteria had no significant difference between WT-MEFs and Atg5-/-MEFs, and RAPA treatment could significant decrease bacterial quantity in WT-MEFs. The number of bacteria in Atg5-/-MEFs was highest among cells infected by ST8-△pRsT98; however, bacterial quantity in RAPA-treatment WT-MEFs had no significant difference than in WT-MEFs without RAPA treatment infected by ST8-△pRST98.2. The effect of spvB on cellular autophagy and its’possible molecular mechanism.(1) Relative quantity of LC3-Ⅱ protein expression in infected cells. Expression of LC3-Ⅱ protein in WT-MEFs infected by SR-11strain was significant weaker than in SR-11-AspvB strain-infected WT-MEFs at1h and3h. Infected cells did not express LC3-Ⅱ protein during laterly infection from5h to10h.(2) Relative quantity of PLD1and PLD2protein expression in infected cells. The expression of PLD1protein in SR-11strain-infected cells was higher than in cells stimulated by SR-11-AspvB strain, while PLD2protein expression had no significant difference between in two kinds of bacterial-infected cells.(3) Relative quantity of TLR4protein expression in infected cells. The intensity of TLR4protein expression in SR-11strain-infected cells was significant lower than in cells infected by SR-11-△spvB strain at each time points of infection.(4) PLD activity of infected cells. The activity of PLD in cells infected by SR-11and SR-11-△spvB strain was both higher than the basic PLD activity of uninfected cells. PLD activity in SR-11strain-infected cells was higher than in cells infected by SR-11-△spvB strain at each time points of infection. PLD activity of infected cells reached climax at1h and gradually declined at lately infection.(5) TLR4mRNA expression of infected cells. The expression of TLR4mRNA in SR-11strain-infected cells was weaker than in cells infected by SR-11-△spvB strain at each time points of infection.(6) The apoptotic rate of infected cells. The apoptotic rate in WT-MEFs infected by SR-11-△spvB strain was significant lower than in SR-11strain-infected WT-MEFs and SR-11-△spvB strain-infected Atg5-/-MEFs, while the apoptotic rate had no significant difference between in WT-MEFs and Atg5-/-MEFs infected by SR-11strain.(7) Cytokines secretion of infected cells. Expression of Th1type cytokine such as IFN-y in SR-11strain-infected cells was weaker than in cells infected by SR-11-△spvB strain from1h to5h, while the secretion of Th2type cytokines such as IL-4and IL-13in cells infected by SR-11strain were higher than in cells infected by SR-11-△spvB. Three cytokines had no significant difference between in cells infected by two kinds of bacteria at8h and10h.(8) Bacterial quantity of infected cells. The number of intracellular bacterial quantity in SR-11-△spvB strain-infected WT-MEFs was significant lower than in WT-MEFs infected by SR-11and Atg5-/-MEFs infected by SR-11-△spvB strain. Intracellular bacterial quantity had no significant difference between in WT-MEFs and Atg5-/-MEFs both infected by SR-11strain.Conclusions:1. Salmonella virulence plasmid pRST98can promote intracellular bacte(?)al survival or proliferation through inhibiting cellular autophagy and consequently enhance apoptosis of infected fibroblasts, a mechanism underlying pRST98-mediated virulence in S. typhi.2. The capability of autophagy inhibition by pRST98is due to the key virulent segment on its genetic sequence spvB. SpvB can inhibit cellular autophagy through TLR4expression inhibition, PLD1expression promotion and PLD activity enhancement, regulation Th1/Th2shift away through increasing Th2type cytokines IL-4and IL-13secretion and decreasing Th1type cytokine IFN-γ secretion. It is speculated that mTOR activation through serine/threonine protein kinase (AKT)-dependent pathway may be one signaling pathway of autophagy inhibition induced by spvB.3. Rapamycin (mTOR inhibitor) can attenuate autophagy inhibition induced by spv, therefore enhance the competent of intracellular bacteria elimination by autophagy and lessen the injury of host cells.4. Autophagy can act as a "double-edged sword" function in Salmonella infection. Moderate activation of autophagy has a beneficial effect on restricting Salmonella proliferation and reducing the injury of host cells, while its deficiency, inhibition or over enhancement can worse infection and result in the more serious damage of host cells.更多还原