核苷二磷酸激酶调节铜绿假单胞菌毒力及致病性的机制研究

发布时间：2018-01-04 19:16

  本文关键词：核苷二磷酸激酶调节铜绿假单胞菌毒力及致病性的机制研究　出处：《第三军医大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 铜绿假单胞菌 核苷二磷酸激酶 胞外毒力因子 Ⅲ型分泌系统 群体感应系统 毒力 宿主致病性

【摘要】：铜绿假单胞菌(Pseudomonas aeruginosa,PA)又称绿脓杆菌,是引起烧创伤、免疫功能低下及肺纤维化等病人菌血症、尿道炎或肺炎等医院内获得性感染最常见的条件性致病菌之一。PA具有目前已知的最大细菌基因组(6.3 million base pairs,6.3Mbp),其开放阅读框(Open Reading Frame,ORF)多达5,570个。目前,已鉴定或预测具有功能的ORFs仅占总ORFs的54.2%,其中包括参与蛋白分泌系统、二组分调节系统、外膜蛋白形成、物质代谢/转运、抗生素抵抗及细菌表面分子形成等多种功能的ORFs。PA庞大而复杂的基因组结构,为其适应严苛的宿主环境并引发感染奠定了重要的分子基础。在PA众多ORFs中,与蛋白分泌相关ORFs在调节PA毒力及致病性中起到关键作用。PA具有五大蛋白分泌系统:I-Ⅲ型和V-VI型。I型蛋白分泌系统(type I secretion system,T1SS)和II型蛋白分泌系统(type II secretion system,T2SS)可将蛋白直接分泌至细菌胞外。其中由T1SS或T2SS分泌的碱性蛋白酶、弹性蛋白酶(Las B、Las A)、磷脂酶C、碱性磷酸酶可促进组织损伤;由T2SS分泌的脂酶可促进脂肪水解。Ⅲ型蛋白分泌系统(type Ⅲ secretion system,T3SS)负责将具有生物学活性的效应蛋白Exo S、Exo T(均具有ADP-核糖基转移酶活性和Rho GTPase激活蛋白活性)、Exo Y(腺苷酸环化酶活性)和/或Exo U(磷脂酶活性)注射入宿主细胞,进而影响细胞凋亡、增殖、吞噬和炎症等一系列功能;而T5SS和T6SS则通过分泌酯酶、血细胞凝激素和磷脂酶D等毒力蛋白/因子促进对组织的损伤及红细胞凝集。此外,PA可通过促进绿脓素、绿色荧光素(载铁体)、藻酸盐等物质合成、调节细菌运动(如:集群运动)及促进生物膜形成等方式促进PA对宿主的致病性。目前,调节PA毒力及致病性相关机制已有诸多报道,但由于这些机制的复杂性和调节方式的多样性,对其深入的研究仍具有重大创新性和挑战性。核苷二磷酸激酶(nucleoside diphosphate kinase,Ndk)是调节原核和真核生物核酸代谢的重要激酶。尽管Ndk对促进PA藻酸盐的合成及调节宿主细胞的吞噬、炎症应答等作用已有报道,但其对PA毒力及致病性的系统性影响及相应机制仍不清楚。本课题组前期预实验发现,在PA急性肺感染小鼠模型中,ndk基因表达水平显著降低并伴随着T3SS基因表达的上调,提示Ndk是PA急性期感染中的一种重要宿主应答蛋白,可能通过自身表达水平的改变,调控PA毒力及对宿主的致病性。因此,本文通过构建ndk基因敲除菌株及回补菌株,分别在小鼠肺部感染模型和细胞感染模型中,考察Ndk在调节PA宿主致病性及细胞毒性中的作用及可能机制。此外,通过转录组测序,考察ndk基因敲除对PA全基因组基因表达的影响;采用RT-q PCR、Western blot等方法及相应表型鉴定,验证转录组测序结果,并进一步探讨Ndk在调节PA群体感应(quorum sensing,QS)系统、胞外毒力因子产生及T3SS中的作用及可能机制。本实验主要的研究内容及结果如下:Ndk在调节PA对宿主致病性中的作用。采用Red重组酶系统构建PAO1和各PAO1基因突变菌株,并将其滴鼻感染小鼠构建小鼠肺感染模型,动态检测小鼠死亡率、肺组织水肿、病变及炎症等情况;将PAO1和各PAO1基因突变菌株感染A549细胞株,通过CCK8、免疫荧光染色、流式细胞术和Western blot等方法,检测细胞的活性、凋亡、膜通透性及T3SS效应蛋白的细胞内转运情况。结果显示,小鼠急性期肺部感染时,ndk基因敲除通过上调T3SS相关蛋白表达水平,加重了由T3SS介导的小鼠肺组织损伤、炎症应答,并增加了小鼠死亡率;ndk基因敲除通过上调T3SS相关蛋白表达水平或增加T3SS效应蛋白转运促进了PA对细胞的毒性、诱导了细胞凋亡或增加了细胞膜通透性。Ndk对PA全基因组基因表达的影响。通过转录组测序检测了PAO1及△ndk菌株基因表达差异。通过GO(gene ontology)功能注释和KEGG(kyoto encyclopedia of genes and genomes)通路富集分析等生物信息学方法,考察Ndk对PA全基因组基因表达的影响。结果显示,ndk基因敲除影响了PA物质代谢/转运等基础生命活动相关基因表达。此外,ndk基因敲除影响了T3SS、胞外毒力因子合成等细菌毒力相关的基因表达。Ndk对PA毒力相关表型的影响。采用RT-q PCR、Western blot方法及相应表型鉴定,检测了PAO1、△ndk和△ndk+(ndk基因回补株)胞外毒力因子和T3SS相关的基因及蛋白表达情况、运动及生物膜形成能力。结果显示,ndk基因敲除抑制了胞外毒力因子如:弹性蛋白酶、碱性磷酸酶、吩嗪等相关基因表达水平及弹性蛋白酶、绿脓素合成。然而,ndk基因敲除上调了T3SS基因及蛋白的表达水平。此外,ndk基因敲除抑制了PA集群运动及生物膜形成能力。Ndk对PA QS系统的调节作用。采用RT-q PCR、信号分子报告菌株平板法及高效液相色谱方法,检测PAO1、△ndk和△ndk+菌株三大QS系统(las、rhl及pqs系统)信号分子合成酶和转录因子相关基因的基因表达水平及信号分子产量。结果显示,ndk基因敲除抑制了QS系统信号分子合成酶基因(las I、rhl I和pqs A)和转录因子基因(las R、rhl R和pqs R)的基因表达及信号分子(3-oxo-C12-HSL、C4-HSL和PQS)合成。Ndk通过QS系统调节PA毒力相关表型。通过在△ndk菌株培养基中添加外源性信号分子3-oxo-C12-HSL、C4-HSL或PQS,检测△ndk菌株胞外毒力因子和T3SS相关基因的基因及蛋白表达水平。结果显示,添加C4-HSL促进了△ndk菌株弹性蛋白酶及绿脓素的合成;而添加C4-HSL或PQS抑制了△ndk菌株上调的T3SS基因及蛋白表达水平。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa PA) is also called Pseudomonas aeruginosa, is caused by burn and trauma, low immune function and pulmonary fibrosis in patients with bacteremia, hospital acquired pneumonia, urethritis or opportunistic infections of the most common pathogens of.PA has known the bacterial genome (6.3 million base pairs, 6.3Mbp) the open reading frame (Open, Reading Frame, ORF) as many as 5570. At present, the identified or predicted functional ORFs accounted for only 54.2% of total ORFs, including participation in protein secretion system, two component regulating system, outer membrane protein formation, metabolism / transport, antibiotic resistance and bacterial surface molecules etc. the function of ORFs.PA large and complex genome structure, to adapt to the harsh environment for the host and cause infection laid the important molecular basis. In the PA ORFs, and ORFs in the regulation of PA secretion related protein Has five protein secretion system plays a key role in virulence and pathogenicity of.PA in I-: type III and type V-VI type.I protein secretion system (type I secretion system, T1SS) and type II protein secretion system (type II secretion system, T2SS) can be directly secreted proteins to the bacterial extracellular alkaline protease which secreted. By T1SS or T2SS, elastase (Las B Las A,), phospholipase C, alkaline phosphatase can promote tissue injury; secreted by T2SS lipase can catalyze the hydrolysis of fats. Type III secretion system (type secretion system, T3SS) will be responsible for the biological activity of protein Exo Exo T effect S (have ADP- ribosyltransferase activity and Rho GTPase activity, Exo activated protein) Y (adenylyl cyclase activity) and / or Exo U (phospholipase activity) injected into the host cell, and cell apoptosis, proliferation, phagocytosis and inflammation of a series of functions such as T5SS and T; 6SS is secreted by esterase, blood cells coagulation hormone and phospholipase D virulence factor promotes protein / injury and erythrocyte agglutination of tissue. In addition, PA can promote the pyocyanin, green fluorescein (IRONLOADING body), substances such as alginate synthesis, regulation of bacterial movement (such as: cluster motion) and promote biofilm in order to promote the formation of pathogenic PA on the host. At present, the existing adjustment mechanism of PA virulence and pathogenicity of many reports, but because of the diversity and complexity of these mechanisms regulating mode, still has great innovation and challenge for further study. Two nucleoside phosphate kinase (nucleoside diphosphate, kinase, Ndk) is an important kinase regulation of prokaryotic and eukaryotic organisms. Although Ndk nucleic acid metabolism of phagocytosis promoting the synthesis of PA alginate and regulating host cells, inflammatory response and other effects have been reported, but its virulence and pathogenicity of PA system Effect and mechanism are still unclear. The pre experiment found that in a mouse model of acute lung infection in PA, the expression level of NDK gene decreased significantly accompanied by upregulation of T3SS, suggesting that Ndk is a kind of important host response protein PA in acute stage of infection, may change the expression levels by itself. PA regulation of virulence and pathogenicity to the host. Therefore, this paper constructs NDK gene knockout strains and revertant strains, respectively in the model of pulmonary infection in mice and cell infection model, to observe the role of Ndk in regulating PA pathogenicity and host cell toxicity and the possible mechanism. In addition, through transcriptome sequencing, NDK investigation gene knockout effects on expression of PA gene by RT-q; PCR, Western blot and other methods and the corresponding phenotypic identification, validation transcriptome sequencing results, and to further explore the regulation of Ndk in PA group (quorum sens should be Ing, QS) system, produce extracellular virulence factors and the role of T3SS and the possible mechanism. The main research contents and results of this experiment are as follows: Ndk in the regulation of PA on host pathogenic role in the construction of PAO1 and PAO1. The gene mutation strains using Red recombination system, and the intranasal infection of mice construction of mouse lung infection model, dynamic detection of mouse mortality, pulmonary edema, inflammatory disease and other conditions; the PAO1 and the PAO1 gene mutation in A549 cells infected by CCK8 strain, immunofluorescence staining, flow cytometry and Western blot methods, detection of cell activity, apoptosis, membrane permeability and T3SS transport the effect of protein in cells. The results showed that the acute phase of pulmonary infection in mice, NDK knockout expression through upregulation of T3SS related protein, exacerbated the lung injury in mice by T3SS mediated inflammatory response, and increase the mortality of mice; NDK gene knockout through upregulation of T3SS protein expression or increased T3SS protein transport and promote the effect of PA on cell toxicity, effects of cell apoptosis or increase the cell membrane permeability of.Ndk on the expression of PA gene. Through transcriptome sequencing to detect the differential expression of PAO1 and NDK strains by GO delta gene. (Gene Ontology KEGG (Kyoto) functional annotation and Encyclopedia of genes and genomes) pathway enrichment analysis by bioinformatics method, the effects of Ndk on the expression of PA gene. The results showed that the effect of NDK gene expression in PA metabolism / transport basis of life activities related genes. In addition, effects of NDK gene knockout T3SS, bacterial virulence genes related to extracellular virulence factor synthesis influence.Ndk expression of PA virulence related phenotype. Using RT-q PCR, Western blot method and the corresponding phenotypic identification, detection of PA O1, delta NDK and delta ndk+ (NDK gene complementation strain) extracellular virulence genes and proteins associated with T3SS expression, movement and biofilm forming ability. The results showed that NDK gene knockout inhibited extracellular virulence factors such as elastase, alkaline phosphatase, phenazine related gene expression level and elastase pyocyanin, synthesis. However, NDK knockout upregulation of T3SS mRNA and protein expression level. In addition, NDK gene knockout inhibited PA clusters and biofilm formation ability of.Ndk to regulate PA QS system. Using RT-q PCR, detection of PAO1 signal sub report strain plate method and high performance liquid chromatography the chromatographic methods, delta NDK and delta ndk+ strain three QS system (LAS, RHL and PQS) signaling molecules and transcription factor related genes synthetase gene expression level and signal molecular yield. The results showed that NDK gene knockout suppressed QS signal system The son (Las I, RHL synthase gene I and PQS A) and transcription factor genes (Las R, RHL R and PQS R) gene expression and signal molecules (3-oxo-C12-HSL, C4-HSL and PQS) synthesis of.Ndk regulation PA virulence related phenotype by QS system. Through the cultivation of exogenous signal molecule in 3-oxo-C12-HSL matrix in NDK strain, C4-HSL or PQS, the expression level of gene and protein related gene detection of delta NDK strains extracellular virulence factors and T3SS. The results showed that the addition of C4-HSL promoted the synthesis of delta NDK strains and elastase pyocyanin; and the addition of C4-HSL or PQS inhibited T3SS gene and protein in NDK strain upregulates the expression level.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378
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本文编号：1379743