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尿路致病性大肠埃希菌毒力因子tcpc在其免疫逃逸中的作用及致病机制研究

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中华微生物学和免疫学杂志 2024年第3期44卷 198-204页

作者：蒋委余范佳颖樊力铭王家栋江子妍管子瑜欧茜方佳琪浙大城市学院医学院临床医学系杭州310015 浙江大学医学院基础医学系杭州310058

目的探究尿路致病性大肠埃希菌(uropathogenicEscherichia coli,UPEC)毒力因子tcpc在其免疫逃逸中的作用,并初步了解其相关致病机制。方法从C57BL/6小鼠的尿道插管至膀胱,滴注分泌tcpc的UPEC CFT073野生株(CFT073wt)或tcpc基因敲除株(CF... 详细信息

目的探究尿路致病性大肠埃希菌(uropathogenicEscherichia coli,UPEC)毒力因子tcpc在其免疫逃逸中的作用,并初步了解其相关致病机制。方法从C57BL/6小鼠的尿道插管至膀胱,滴注分泌tcpc的UPEC CFT073野生株(CFT073wt)或tcpc基因敲除株(CFT073Δtcpc)悬液109菌落形成单位,构建肾盂肾炎小鼠模型。5 d后处死小鼠取肾脏,观察大体形态变化。HE染色观察肾脏组织病理变化,免疫组织化学法对肾组织中tcpc进行定位。采集感染后的小鼠尿液,十倍稀释法计数尿液中细菌载量,PCR检测小鼠肾脏组织和尿液细菌基因组DNA中的tcpc基因。实时荧光定量PCR检测CFT073wt菌株感染树突状细胞后胞内tcpc mRNA水平。Western blot和ELISA分别检测CFT073wt和CFT073Δtcpc菌株对树突状细胞NF-κB活化和促炎因子水平的影响,激光共聚焦显微镜观察UPEC菌株感染树突状细胞后的细菌活力。结果与CFT073Δtcpc组相比,CFT073wt组小鼠肾脏有明显脓肿产生,肾脏组织中有大量中性粒细胞浸润和tcpc。CFT073wt组小鼠尿液中的细菌载量显著高于CFT073Δtcpc组;PCR结果显示,肾脏组织和尿液中细菌均能扩增出tcpc基因。在CFT073wt菌株感染树突状细胞过程中,胞内tcpc的mRNA和蛋白质水平显著升高。CFT073wt抑制树突状细胞NF-κB信号通路p50的磷酸化水平和促炎因子的产生。tcpc有利于CFT073wt在树突状细胞内的存活。结论tcpc在CFT073wt感染及引发小鼠肾盂肾炎过程中表达水平显著升高,并通过抑制树突状细胞NF-κB信号通路的活化和促炎因子的产生有利于CFT073wt在树突状细胞内的存活,与UPEC致病及抗树突状细胞免疫密切相关。

关键词：免疫逃逸 tcpc 尿路致病性大肠埃希菌肾盂肾炎树突状细胞

tcpc inhibits the phenotypical and functional maturation of dendritic cells

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TcpC inhibits the phenotypical and functional maturation of ...

浙江省免疫学会第十次学术大会

作者： Jie Fang Ruiling Chen Xiaoli Xin Yun Zhou Yujie He Dayong Zhang Baoming Wang Sheng Quan Lihuang Zhang Jianping Pan Department of Clinical Medicine Zhejiang University City College School of Medicine Department of Pathogen Biology Zhejiang University School of Medicine

Objectives:Toll/interleukin 1 receptor domain containing protein encoded by ***(tcpc) was found to be a virulence factor that promotes bacterial survival and is closely related to the pathogenesis of urinary tract *** was demonstrated that tcpc can inhibit bactericidal activity of macrophages by blocking toll-like receptor 4(TLR-4) signaling pathway and plays a crucial role in the pathogenesis of *** aim of the present study is to examine the regulatory effects of tcpc on the phenotypical and functional maturation of dendritic cells(DC).Materials and methods:tcpc-secreting *** strain CFT073 was obtained from ATCC and tcpc-knock out CFT073 mutant strain(CFT073 Atcpc) was generated by X red homologous ***2.4,the C57 BL/6 bone marrow-derived dendritic cell line,was kindly provided by *** *** marrow-derived dendritic cells(BM-DC) were induced in vitro by culturing the C57 BL/6 mice-derived red blood cell-depleted bone marrow cells with GM-CSF and IL-4 and were purified by CD11 c *** dendritic cells were isolated from kidneys from mouse model with *** examine the in vitro regulatory effects of tcpc on dendritic cells,DC2.4 or BM-DC were co-cultured with CFT073 or CFT073 Atcpc in a transwell system for 24 *** of DC and secretion of cytokines by DC were analyzed by FACS and ELISA,***4+ T cells and CD8 T cells were sorted from spleens of OT-I and OT-II mice that were provided by *** *** examine the influence of tcpc on the antigen presentation of DC,CFT073 or CFT073 Atcpc-treated DC2.4 and kidney DC from mouse models with pyelonephritis induced by transurethral instillation of CFT073 or CFT073 Atcpc were co-cultured for72 h with CD4+ T cells or CD8 T cells,at a stimulator to responder ratio of 1:10,in the presence of 2 μg/ml OVA323-339 or *** concentrations of IL-2 and IFN-γ in the culture supernatants were measured by ***:tcpc significantly

关键词： E coli tcpc Dendritic cells Antigen presentation Pyelonephritis

tcpc promotes kidney cells to secrete MIP-2 via p38 MAPK pathway

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TcpC promotes kidney cells to secrete MIP-2 via p38 MAPK pat...

浙江省免疫学会第十次学术大会

作者： Yingzhi Yang Lulu Liu Chong Zhang Jie Fang Dayong Zhang Sheng Quan Baoming Wang Lihuang Zhang Jianping Pan Department of Clinical Medicine Zhejiang University City College School of Medicine

Objective:Toll/interleukin-1 receptor domain containing protein encoded by ***(tcpc) is a virulence factor of some strains of uropathogenic ***(UPEC),and it was found that tcpc promoted bacterial survival and increased the severity of *** is an infection of the upper urinary tract involving bacterial invasion of the renal parenchyma,which is characterized by neutrophil infiltration and abscess ***2,also known as macrophage inflammatory protein 2(MIP-2),is a potent chemoattractant and activator of neutrophils and plays crucial role in the pathogenic process of the disease as *** aim of the present study is to explore the influence of tcpc on the production of MIP-2 by kidney cells,so as to provide further evidence to clarify the pathogenicity of tcpc secreting *** and methods:Human embryonic kidney cell line(HEK-293) was purchased from Shanghai Institute of Biochemistry and Cell *** expressing *** CFT073 strain was obtained from *** tcpc-knock out CFT073 mutant strain(CFT073 Atcpc) was constructed by X red homologous *** models with pyelonephritis were created by transurethral instillation of CFT073 or CFT073 *** from the mouse models were used for histological examination and detection of ***-293 cells were separately co-cultured with different doses of CFT073 or CFT073 Atcpc in transwell *** supernatants were harvested and used for detection of MIP-2 and for determination of chemoattractant activity of *** of the CFT073 or CFT073 Atcpc-treated HEK293 cells were extracted and used for examination of signaling pathway associated molecules by western *** of MIP-2 were determined by ELIS A and chemotaxis assay was employed to determine the chemoattractant activity of ***:Histological examination showed that only a few neutrophils were seen in kidneys from mouse models with pyelonephritis caused by infe

关键词： tcpc MIP-2/CXCL2 Neutrophils Pyelonephritis Signaling pathway

tcpc is not only an E3 ubiquitin ligase but also an inhibitor of USP14 deubiquitinase

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TcpC is not only an E3 ubiquitin ligase but also an inhibito...

浙江省免疫学会第十三次学术大会

作者： Jiaqi Fang Qian Ou Xinpeng Ma Weiwei Luo Wenting Li Sisi Li Jiajie Jing Ziyan Jiang Jiadong Wang Weiyu Jiang Jianping Pan Institute of Translational Medicine Hangzhou City University Department of Clinical Medicine Hangzhou City University School of Medicine Department of Basic Medical Sciences Zhejiang University School of Medicine

Background:Toll/interleukin 1 receptor domain containing protein encoded by ***（tcpc） is a multifunctional virulence factor in the majority of uropathogenic ***（UPEC） strains and blocks the TLR signaling pathway by serving as an E3 ubiquitin ligase that promotes MyD88 degradation by ubiquitin-proteasome pathway,hereby inhibiting the TLR mediated activation of ***:This study aims to investigate if tcpc can also inhibit the activity of deubiquitinase and its related *** and methods:tcpc secreting wild-type CFT073(CFT073) was kindly provided by Professor Jian-guo *** knock out CFT073 mutant（CFT073Δtcpc） was constructed in our *** human monocytic cell line THP-1 and mouse macrophage cell lines J774 A.1 were obtained from Shanghai Institute of Biochemistry and Cell *** situ co-localization of tcpc with USP14,USP14 with ubiquitin and Rpn1 in kidneys from pyelonephritis mouse model was determined by confocal *** of UPEC in macrophages was observed by holographic 3 D live cell imaging system,flow cytometry and confocal *** of deubiquitinase and proteasome were measured by fluorescent microplate reader.K48-ubiquitination of MyD88 in rtcpc treated macrophages was detected by *** mRNA and protein expression levels of pro-inflammatory cytokines were detected by qRT-PCR and *** abilities of rtcpc with rUSP14 was determined by surface plasmon resonance（SPR） and microscale thermophoresis（MST）.Transcription and protein level of ubiquitin and proteasome related genes were detected by transcriptome and proteome *** amino acids required to maintain the deubiquitinase inhibitory activity in rtcpc were identified by ***:In situ co-localization of tcpc and ubiquitin with USP14 or PSMD2 with USP14 was significantly enhanced or declined respectively in kidney from CFT073 infected mice compared to kidney isolated from mice infect

关键词： tcpc inhibit deubiquitinase USP14 innate immune

tcpc secreting uropathogenic *** promoted kidney cells to secrete MIP-2 via p38 MAPK pathway

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TcpC secreting uropathogenic *** promoted kidney cells to se...

第十一届全国免疫学学术大会

作者： He Yujie Zhang Chong Fang Jie Yang Yingzhi Zhang Dayong Wang Baoming Wang Linyao Pan Jianping School of Medicine Zhejiang University City College

Background: Toll/interleukin-1 receptor domain containing protein encoded by E. coli(tcpc) is an important virulence factor in most strains of uropathogenic Escherichia coli(UPEC) and inhibits the macrophage-mediated innate immunity, which plays crucial role in the pathogenesis of pyelonephritis. Objectives: To examine the regulatory effects of tcpc on production of MIP-2 in kidney cells and its related mechanism. Methods: Mouse models with pyelonephritis were established by transurethral instillation of tcpc-secreting wild type UPEC CFT073(tcpcwt) or tcp C-knock out CFT073(tcpc-/-). HEK-293 cells were co-cultured with tcpcwt or tcpc-/- in transwell. MIP-2 concentrations were measured by ELISA. Signaling transduction proteins were analyzed by Western blot. Results: MIP-2 concentration in kidney homogenates from tcpcwt caused murine pyelonephritis models was significantly higher than that from tcpc-/- caused models. In vitro, tcpcwt dose-dependently promoted MIP-2 secretion by HEK-293 cells. Concentration of MIP-2 in culture supernatants of HEK-293 co-cultured with tcpcwt was profoundly higher than that co-cultured with tcpc-/-. In the presence of anti-tcpc antibody, this enhancement effect of tcpcwt on MIP-2 production was completely abrogated. tcpc-/- treatment had no effect on p38 MAPK pathway, while tcpcwt treatment resulted in the activation of p38 MAPK in HEK-293 cells. Inhibition of p38 MAPK could significantly decrease MIP-2 concentration and neutrophil recruitment activity in the supernatants of HEK-293 cells co-cultured with tcpcwt. Conclusion: tcpc could promote kidney cells to produce MIP-2 through p38 MAPK pathway, which might contribute to the characteristic histological change of pyelonephritis.

关键词： Uropathogenic E.coli Pyelonephritis tcpc MIP-2

尿路致病性大肠埃希菌通过tcpc抑制巨噬细胞吞噬的信号通路研究

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中华微生物学和免疫学杂志 2022年第8期42卷 602-608页

作者：马欣鹏罗薇薇李雯婷欧茜裘佳灵岑诺方佳琪浙大城市学院医学院杭州310015

目的探究尿路致病性大肠埃希菌tcpc N端泛素连接酶片段抑制巨噬细胞吞噬的信号通路。方法生物信息学软件分析tcpc N端泛素连接酶片段氨基酸序列结构与功能,并预测其功能位点。PCR扩增tcpc N端不同长度片段tcpc-330、tcpc-450和tcpc-510... 详细信息

目的探究尿路致病性大肠埃希菌tcpc N端泛素连接酶片段抑制巨噬细胞吞噬的信号通路。方法生物信息学软件分析tcpc N端泛素连接酶片段氨基酸序列结构与功能,并预测其功能位点。PCR扩增tcpc N端不同长度片段tcpc-330、tcpc-450和tcpc-510基因并构建其原核表达系统。Ni-NTA亲和层析法纯化目的重组蛋白rtcpc-N110、rtcpc-N150和rtcpc-N170;Detoxi-Gel层析柱去除目的重组蛋白中可能含有的LPS。Western blot和实时荧光定量RT-PCR检测rtcpc-N110、rtcpc-N150、rtcpc-N170和rtcpc-TIR分别孵育巨噬细胞后胞内MyD88蛋白水平和mRNA水平。Western blot检测上述蛋白质孵育巨噬细胞后NF-κB信号通路活化情况。ELISA检测上述蛋白质孵育巨噬细胞后促炎因子水平。结果tcpc N端片段氨基酸序列中第12位半胱氨酸、第104位色氨酸和第106位色氨酸是其泛素连接酶活性的功能位点。所构建的原核表达系统在IPTG诱导下能够有效表达rtcpc-N110、rtcpc-N150和rtcpc-N170,Ni-NTA亲和层析法提纯后均仅见单一蛋白质条带。Detoxi-gel亲和层析柱处理后rtcpc-N110、rtcpc-N150和rtcpc-N170中LPS为阴性。tcpc泛素连接酶片段能够抑制MyD88蛋白水平但不影响其mRNA水平。tcpc泛素连接酶片段显著抑制LPS诱导的NF-κB信号通路p50和p65的磷酸化。tcpc泛素连接酶片段显著抑制LPS诱导的巨噬细胞促炎因子的产生。结论rtcpc-N110、rtcpc-N150和rtcpc-N170具有抑制MyD88蛋白水平和NF-κB信号通路活化功能,与抑制巨噬细胞固有免疫活性密切相关。

关键词：尿路致病性大肠埃希菌 tcpc 泛素连接酶抑制固有免疫

tcpc对肾盂肾炎模型小鼠肾脏DCs分化成熟的影响

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TcpC对肾盂肾炎模型小鼠肾脏DCs分化成熟的影响

作者：陈瑞玲浙江大学

学位级别：硕士

背景和目的泌尿道感染(urinarytractinfection,UTI)是临床上最常见的感染性疾病之一,主要由细菌自尿道逆行所致,易复发。部分重症患者可导致肾功能衰竭。有研究表明,导致泌尿道感染的病原菌近80%为大肠埃希菌。尿路致病性大肠埃希菌CFT... 详细信息

背景和目的泌尿道感染(urinarytractinfection,UTI)是临床上最常见的感染性疾病之一,主要由细菌自尿道逆行所致,易复发。部分重症患者可导致肾功能衰竭。有研究表明,导致泌尿道感染的病原菌近80%为大肠埃希菌。尿路致病性大肠埃希菌CFT073菌株可分泌一种含有Toll/interleukin 1 receptor结构域的蛋白(tcpc),与大肠埃希菌所致肾盂肾炎的发生发展密切相关。Toll样受体(TLR)是一类主要表达于巨噬细胞、DCs等抗原提呈细胞的模式识别受体,可识别和结合相应的病原相关分子模式,激活信号传导,介导免疫应答。tcpc可直接与MyD88结合,阻断TLR信号通路,抑制NF-κB的激活,抑制巨噬细胞的功能,改变宿主固有免疫应答功能状态,从而增强细菌的毒力,在大肠埃希菌所致肾盂肾炎中发挥重要作用。在感染性疾病中,机体通过免疫系统来识别和清除入侵的病原微生物,其过程十分复杂,包括固有免疫和适应性免疫。树突状细胞(dendriticcells,DCs)是迄今所知功能最强的专职抗原提呈细胞,在免疫应答的首要环节即抗原提呈中起重要作用。未成熟DCs具有极强的抗原摄取、处理和加工能力,而成熟DCs具有很强的抗原提呈功能,主要参与细胞免疫和T细胞依赖的体液免疫应答。同时,DCs又是固有免疫和适应性免疫的桥梁和纽带。DCs的免疫调节作用与其表面诸多受体分子(如TLRs和C型凝集素)相关。TLRs是重要的模式识别受体,在DCs分化成熟中发挥重要作用。TLR可特异性识别细菌、虫体、真菌和病毒等保守的PAMPs而捕获病原体,调控DCs成熟和细胞因子分泌,从而调节抗原提呈、T细胞的激活,影响和控制固有免疫和适应性免疫应答的强度和质量。而肾小管间质内分布大量的DCs,可通过调节固有免疫和适应性免疫应答而清除病原体,影响肾盂肾炎的发生和发展,在肾损伤和肾炎中均起重要作用。因此,研究tcpc对肾盂肾炎模型小鼠肾脏DCs分化成熟的影响,为阐明大肠埃希菌所致急性肾盂肾炎的病理生理机制提供新的实验依据。方法1肾盂肾炎小鼠模型的建立腹腔注射三澳乙醇麻药,麻醉小鼠;用弯有35。角的留置针经尿道插入膀胱,注入0.1 ml浓度为1011/ml的菌液或生理盐水,3小时后第二次注射,3天后处死模型小鼠,取肾脏用于后续分析。本实验设为3组:NS组,注入生理盐水;tcpcwt组,注入野生型CFT073菌株;tcpcdel组,注入tcpc基因敲除的CFT073菌株。2石蜡切片的制作与HE染色取材固定,冲洗,脱水,透明,浸蜡,包埋,切片,贴片,拷片。HE染色:脱蜡,细胞核染色(苏木精染色10min;自来水洗;盐酸酒精3-5 s;自来水洗),细胞质染色(伊红染色15-20 min;70%酒精;80%酒精),脱水,透明,封片。3免疫磁珠分选肾脏DCs取小鼠肾脏,剪碎,加入消化液消化25 min,过滤网,离心,加入5 ml Tris-NH4Cl,常温5-10 min,离心重悬得细胞悬液;细胞计数,加入少许Buffer(PBS+0.5%FBS+2mMEDTA)洗涤,离心,弃上清,每108个细胞用100μl Buffer重悬,每108个细胞加入100μl的CD11c+磁珠抗体体4℃孵育育2 min,每107个细胞加入1-2 ml Buffer洗涤,离心,弃上清,重悬;磁珠分选:将MACS分离磁铁安装到MACS多用支架上,并将MS分选柱放到磁铁中,润洗分选柱,将细胞悬液上样到分选柱上;用缓冲液冲洗分离柱,每次0.5 ml,洗3次;取下分选柱,用lmlBuffer洗脱柱子内的细胞,即为所要的DCs。4肾脏DCs表型变化的检测流式细胞术分析DCs表面分子CD40、CD54、CD80、CD86、MHC-Ⅰ 及 MHC-Ⅱ 的表达。5 DC细胞培养将分选的各组肾脏DCs培养于RPMI-1640培养基(含10%FBS,100 U/ml青链霉素混合液),细胞配成5×105 cells/ml,铺于24孔板,每孔1 ml。于37℃含5%CO2的孵箱内培养48 h后收集细胞上清液。6免疫磁珠分选OT-Ⅰ和OT-Ⅱ小鼠脾脏中CD4T及CD8T细胞取小鼠脾脏用毛玻璃磨碎,过滤网,离心,加Tris-NH4Cl裂解红细胞,离心,重悬后计数,加入磁珠抗体,孵育15 min过柱,洗柱之后将MS柱撤离磁场,洗脱所要的T细胞。7 tcpc对DC抗原提呈功能的影响(1)DCs分别与大肠杆菌CFT073野生株和tcpc基因敲除株共培养收集DC2.4,洗涤后配成3×105 cells/ml铺于Transwell24孔板的下室,1 ml/孔;上室加3×106个细菌(细胞数:细菌数=1:10)。以3种方式处理:空白对照组,即DC2.4组;DC2.4+tcpcwt组;DC2.4+tcpcdel组。每组做3个复

关键词：肾盂肾炎树突状细胞 tcpc 大肠埃希菌抗原提呈

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tcpc调控树突状细胞作用机制的研究

TcpC调控树突状细胞作用机制的研究

作者：何玉洁浙江大学

学位级别：硕士

背景和目的树突状细胞(dendritic cells,DCs)是迄今为止体内功能最强大的专职抗原提呈细胞,广泛分布于外周组织中。正常情况下体内绝大多数DC都处于未成熟状态,未成熟的DC可通过其表面受体介导的内吞作用、巨吞饮作用以及吞噬作用等方... 详细信息

背景和目的树突状细胞(dendritic cells,DCs)是迄今为止体内功能最强大的专职抗原提呈细胞,广泛分布于外周组织中。正常情况下体内绝大多数DC都处于未成熟状态,未成熟的DC可通过其表面受体介导的内吞作用、巨吞饮作用以及吞噬作用等方式摄取抗原,在摄取抗原后或受到某些刺激(如炎性信号LPS、IL-1β、TNF-α)后,未成熟的DC即开始分化成熟。DC的抗原提呈功能可直接或间接激活T、B细胞,因此DC在免疫应答中占据独特地位,对DC的深入研究有助于我们了解机体免疫应答的发生和调控机制,对肿瘤、移植排斥、感染、自身免疫病等发生发展机制及治疗也具有重要的理论意义和实际意义。Toll样受体(Toll like receptors,TLRs)是一类结构功能较为明确的模式识别受体,在多种组织和细胞中广泛分布,并且在一些固有免疫细胞如巨噬细胞、DCs中表达较高,TLRs可以通过识别并结合病原体的相关模式分子(pathogen associated molecular pattern,PAMP)来活化免疫细胞并表达一系列的免疫效应分子,在免疫应答和炎症反应中发挥重要作用。尿路感染(urinary tract infection,UTI)是由各种病原体入侵泌尿系统、侵犯尿路黏膜或者尿路组织而引发的尿路炎症,临床特征一般表现为腰痛、尿频、尿急、尿痛,引起泌尿系统炎症的致病菌大部分为肠道的大肠埃希菌。针对尿路感染目前多采用抗生素类药物进行治疗,但随着病原菌耐药性的增加给尿路感染的治疗带来了极大挑战。tcpc是由尿路致病性大肠埃希菌CFT073菌株分泌的一种含有TIR(Toll/interleukin-1 receptor)结构域的蛋白,因其TIR结构域与TLR的TIR结构域结构相似,因此tcpc可以直接与TLR信号通路的接头蛋白髓样分化因子 88(Myeloid differentiation factor 88,MyD88)结合,从而阻断 TLR 信号通路,干扰机体正常的免疫应答。目前关于tcpc干扰机体免疫应答的研究多集中于巨噬细胞、中性粒细胞以及上皮细胞,尚无tcpc对树突状细胞分化和功能影响的研究报道。本课题组的前期研究发现,tcpc能够抑制DC的分化成熟以及MHC(maj or histocompatibility complex,MHC)Ⅱ类分子途径对抗原的提呈。本课题拟研究tcpc调控树突状细胞的作用机制,旨在为进一步阐明tcpc介导的免疫逃逸机制提供新的实验依据。方法1 tcpc蛋白的诱导表达冻融已鉴定好的含tcpc基因片段的工程菌*** BL21pET42a-tcpc并划线接种于含卡那霉素的LB固体培养基中,37℃培养箱中培养过夜后从平板上挑取单个分离良好的、直径约为2-3mm的白色半透明菌落于含卡那霉素的LB液体培养基中,37℃220rpm振荡培养过夜。吸取过夜后的菌液1ml并加至含卡那霉素的LB液体培养基中,37℃ 220rpm振荡培养至OD值为0.6-0.8,加入IPTG,37℃220rpm振荡培养,诱导tcpc蛋白的表达。2 tcpc蛋白的纯化浓缩采用镍柱分离纯化、超滤管浓缩的方法。3兔抗tcpc多克隆抗体的制备将浓缩后的tcpc蛋白与弗氏佐剂及弗氏不完全佐剂等量混合乳化至"油包水"状态,初次免疫之前要先进行耳缘静脉采血作为阴性对照,初次免疫之后的第三周开始加强免疫,每周一次共三次。末次免疫10天后先采少量血进行效价测定。4兔抗tcpc多克隆抗体效价的测定采用双向琼脂扩散的方法进行测定。5兔抗tcpc多克隆抗体的纯化效价达到要求后,对家兔进行颈动脉采血,收集获得的血液并分离血清,再采用饱和硫酸铵法纯化血清中的免疫球蛋白IgG。6兔抗tcpc多克隆抗体的特异性检测Western blotting检测制备的抗体对已纯化的tcpc蛋白以及CFT073菌株分泌的tcpc天然蛋白的特异性,同时用tcpc基因敲除株tcpc-/-作为对照。7检测DCs能否摄取tcpc蛋白收集DC2.4,洗涤后配成3×105 cells/ml铺于Transwell 24孔板的下室,1ml/孔;上室加3×106个细菌(MOI=细菌数:细胞数=10:1)。以3种方式处理:空白对照组,即DC2.4组;DC2.4+tcpcwt组;DC2.4+tcpc-/-组,每组做3个复孔。在不同的时间点收集细胞,裂解细胞并获取胞内蛋白,用制备的兔抗tcpc多克隆抗体进行western blotting检测。8检测tcpc蛋白进入DCs胞内后的作用机制收集DC2.4,洗涤后配成3×105 cells/ml并铺于Transwell 24孔板的下室,1ml/孔;上室加3×106个细菌(MOI=细菌数:细胞数=10:1)。以3种方式处理:空白对照组,即DC2.4组;DC

关键词： tcpc 树突状细胞 MyD88蛋白 p65蛋白磷酸化 TLR信号通路

尿路致病性大肠杆菌毒力因子tcpc对中性粒细胞NETosis的影响及其机制研究

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尿路致病性大肠杆菌毒力因子TcpC对中性粒细胞NETosis的影响及其...

作者：欧茜浙江大学

学位级别：硕士

背景和目的:尿路感染(urinary tract infection,UTI)是由各种病原体入侵泌尿系统、侵犯尿路黏膜或者尿路组织而引起的尿路炎症,部分严重患者可导致肾功能衰竭。大肠埃希菌(Escherichia coli,***)是尿路感染最常见的病原体,约80%的UTI由... 详细信息

背景和目的:尿路感染(urinary tract infection,UTI)是由各种病原体入侵泌尿系统、侵犯尿路黏膜或者尿路组织而引起的尿路炎症,部分严重患者可导致肾功能衰竭。大肠埃希菌(Escherichia coli,***)是尿路感染最常见的病原体,约80%的UTI由***引起。尿路致病性大肠埃希菌(uropathogenic ***,UPEC)可以特异性地粘附、植入尿路黏膜上皮细胞,导致尿路上行感染。UPEC可分泌一种含有Toll/白细胞介素1受体(Toll/interleukin 1 receptor,TIR)结构域的蛋白(TIR containing protein encoded by ***,tcpc),它可以直接与Toll样受体(Toll-like receptor,TLR)信号通路中的关键接头蛋白髓样分化因子88(myeloid differentiation factor 88,My D88)结合,阻断TLR介导的信号途径,从而抑制巨噬细胞的活化和杀菌功能,利于UPEC在体内的存活。本实验室前期发现tcpc是一种多功能毒力因子,除了可通过诱导肾细胞产生巨噬细胞炎性蛋白-2(macrophage inflammatory protein?2,MIP-2)趋化中性粒细胞浸润,还具有E3泛素连接酶功能,可促进My D88蛋白降解以介导固有免疫逃逸,提示tcpc在UPEC感染和致病过程中发挥关键作用。中性粒细胞是机体受到感染时发挥免疫功能的一道重要防线,目前许多研究发现产生中性粒细胞胞外捕捉陷阱(neutrophil extracellular traps,NETs)是中性粒细胞一种关键的抗感染方式,NETs形成的过程简称为NETosis。中性粒细胞在机体受到病原微生物感染时,可以通过产生NETosis捕捉微生物,然后利用附着在三维网状骨架上的抗菌蛋白杀死微生物。急性肾盂肾炎的特征性病理变化是中性粒细胞浸润,tcpc对中性粒细胞NETosis到底有何影响尚无研究报道。本课题研究tcpc对中性粒细胞NETosis的影响及相关分子机制,旨在为进一步阐明tcpc介导的固有免疫逃逸机制,提供新的实验依据。方法:膀胱内滴注分泌tcpc的CFT073野生株(CFT073)或敲除tcpc基因的CFT073突变株(CFT073),制备C57BL/6小鼠肾盂肾炎模型。采用HE染色和组织免疫荧光染色,观察小鼠肾组织病理学改变以及tcpc对NETosis的影响。用试剂盒分离人外周血中性粒细胞,流式检测细胞纯度后,用1μg/ml LPS诱导人中性粒细胞NETosis,CFT073或CFT073菌株与细胞共培养于Transwell系统中,16 h后制片,用激光共聚焦观察NETosis水平。采用Ni-NTA亲和层析法提取tcpc基因表达的重组蛋白r tcpc。采用鲎试验和Detoxi-gel脂多糖(LPS)亲和层析法分别检测和清除r tcpc中可能污染的LPS。r tcpc处理中性粒细胞210 min后,用激光共聚焦显微镜观察r tcpc进入细胞的方式和中性粒细胞DNA解聚、髓过氧化物酶(Myeloid peroxidase,MPO)表达以及组蛋白瓜氨酸化水平。q RT-PCR和ELISA分别检测r tcpc处理后胞内IL-1β、IL-6和TNF-α的m RNA水平和细胞上清中的分泌水平。激光共聚焦和流式细胞术检测r tcpc处理后NETosis过程中细胞活性氧(reactive oxygen,ROS)水平的变化。全基因转录组测序检测LPS与r tcpc处理后中性粒细胞核内基因转录水平的变化。Western Blot和q RT-PCR检测肽基精氨酸脱氨酶4(peptidylarginine deiminase 4,PAD4)的表达和转录水平。采用生物信息学分析tcpc蛋白的功能结构域以及人和小鼠PAD4的氨基酸序列。采用免疫共沉淀法和体外泛素化试剂盒检测r tcpc处理后PAD4泛素化水平的变化。免疫荧光染色观察r tcpc处理后NETosis过程中PAD4与蛋白酶体标志物PSMD2共定位水平。采用定位突变联合激光共聚焦及Western Blot鉴定r tcpc影响NETosis形成和胞内PAD4蛋白水平的活性位点。结果:tcpc是UPEC重要的毒力因子,能导致肾组织中大量中性粒细胞浸润却显著抑制肾组织原位NETosis。tcpc能通过窖蛋白介导的内吞作用进入细胞且显著抑制LPS诱导的中性粒细胞DNA解聚及MPO、Cit H3、IL-1β、IL-6、TNF-α和ROS的产生。转录组测序结果显示r tcpc可显著抑制NETosis相关基因的转录却促进泛素化相关基因的转录。tcpc处理显著抑制LPS诱导的中性粒细胞胞P

关键词：尿路致病性大肠埃希菌 tcpc 中性粒细胞胞外捕捉陷阱内吞作用肽基精氨酸脱氨酶4 泛素化