發(fā)布時間：2018-01-04 19:16

  本文關(guān)鍵詞：核苷二磷酸激酶調(diào)節(jié)銅綠假單胞菌毒力及致病性的機制研究　出處：《第三軍醫(yī)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 銅綠假單胞菌 核苷二磷酸激酶 胞外毒力因子 Ⅲ型分泌系統(tǒng) 群體感應(yīng)系統(tǒng) 毒力 宿主致病性

【摘要】：銅綠假單胞菌(Pseudomonas aeruginosa,PA)又稱綠膿桿菌,是引起燒創(chuàng)傷、免疫功能低下及肺纖維化等病人菌血癥、尿道炎或肺炎等醫(yī)院內(nèi)獲得性感染最常見的條件性致病菌之一。PA具有目前已知的最大細菌基因組(6.3 million base pairs,6.3Mbp),其開放閱讀框(Open Reading Frame,ORF)多達5,570個。目前,已鑒定或預(yù)測具有功能的ORFs僅占總ORFs的54.2%,其中包括參與蛋白分泌系統(tǒng)、二組分調(diào)節(jié)系統(tǒng)、外膜蛋白形成、物質(zhì)代謝/轉(zhuǎn)運、抗生素抵抗及細菌表面分子形成等多種功能的ORFs。PA龐大而復(fù)雜的基因組結(jié)構(gòu),為其適應(yīng)嚴苛的宿主環(huán)境并引發(fā)感染奠定了重要的分子基礎(chǔ)。在PA眾多ORFs中,與蛋白分泌相關(guān)ORFs在調(diào)節(jié)PA毒力及致病性中起到關(guān)鍵作用。PA具有五大蛋白分泌系統(tǒng):I-Ⅲ型和V-VI型。I型蛋白分泌系統(tǒng)(type I secretion system,T1SS)和II型蛋白分泌系統(tǒng)(type II secretion system,T2SS)可將蛋白直接分泌至細菌胞外。其中由T1SS或T2SS分泌的堿性蛋白酶、彈性蛋白酶(Las B、Las A)、磷脂酶C、堿性磷酸酶可促進組織損傷;由T2SS分泌的脂酶可促進脂肪水解。Ⅲ型蛋白分泌系統(tǒng)(type Ⅲ secretion system,T3SS)負責(zé)將具有生物學(xué)活性的效應(yīng)蛋白Exo S、Exo T(均具有ADP-核糖基轉(zhuǎn)移酶活性和Rho GTPase激活蛋白活性)、Exo Y(腺苷酸環(huán)化酶活性)和/或Exo U(磷脂酶活性)注射入宿主細胞,進而影響細胞凋亡、增殖、吞噬和炎癥等一系列功能;而T5SS和T6SS則通過分泌酯酶、血細胞凝激素和磷脂酶D等毒力蛋白/因子促進對組織的損傷及紅細胞凝集。此外,PA可通過促進綠膿素、綠色熒光素(載鐵體)、藻酸鹽等物質(zhì)合成、調(diào)節(jié)細菌運動(如:集群運動)及促進生物膜形成等方式促進PA對宿主的致病性。目前,調(diào)節(jié)PA毒力及致病性相關(guān)機制已有諸多報道,但由于這些機制的復(fù)雜性和調(diào)節(jié)方式的多樣性,對其深入的研究仍具有重大創(chuàng)新性和挑戰(zhàn)性。核苷二磷酸激酶(nucleoside diphosphate kinase,Ndk)是調(diào)節(jié)原核和真核生物核酸代謝的重要激酶。盡管Ndk對促進PA藻酸鹽的合成及調(diào)節(jié)宿主細胞的吞噬、炎癥應(yīng)答等作用已有報道,但其對PA毒力及致病性的系統(tǒng)性影響及相應(yīng)機制仍不清楚。本課題組前期預(yù)實驗發(fā)現(xiàn),在PA急性肺感染小鼠模型中,ndk基因表達水平顯著降低并伴隨著T3SS基因表達的上調(diào),提示Ndk是PA急性期感染中的一種重要宿主應(yīng)答蛋白,可能通過自身表達水平的改變,調(diào)控PA毒力及對宿主的致病性。因此,本文通過構(gòu)建ndk基因敲除菌株及回補菌株,分別在小鼠肺部感染模型和細胞感染模型中,考察Ndk在調(diào)節(jié)PA宿主致病性及細胞毒性中的作用及可能機制。此外,通過轉(zhuǎn)錄組測序,考察ndk基因敲除對PA全基因組基因表達的影響;采用RT-q PCR、Western blot等方法及相應(yīng)表型鑒定,驗證轉(zhuǎn)錄組測序結(jié)果,并進一步探討Ndk在調(diào)節(jié)PA群體感應(yīng)(quorum sensing,QS)系統(tǒng)、胞外毒力因子產(chǎn)生及T3SS中的作用及可能機制。本實驗主要的研究內(nèi)容及結(jié)果如下:Ndk在調(diào)節(jié)PA對宿主致病性中的作用。采用Red重組酶系統(tǒng)構(gòu)建PAO1和各PAO1基因突變菌株,并將其滴鼻感染小鼠構(gòu)建小鼠肺感染模型,動態(tài)檢測小鼠死亡率、肺組織水腫、病變及炎癥等情況;將PAO1和各PAO1基因突變菌株感染A549細胞株,通過CCK8、免疫熒光染色、流式細胞術(shù)和Western blot等方法,檢測細胞的活性、凋亡、膜通透性及T3SS效應(yīng)蛋白的細胞內(nèi)轉(zhuǎn)運情況。結(jié)果顯示,小鼠急性期肺部感染時,ndk基因敲除通過上調(diào)T3SS相關(guān)蛋白表達水平,加重了由T3SS介導(dǎo)的小鼠肺組織損傷、炎癥應(yīng)答,并增加了小鼠死亡率;ndk基因敲除通過上調(diào)T3SS相關(guān)蛋白表達水平或增加T3SS效應(yīng)蛋白轉(zhuǎn)運促進了PA對細胞的毒性、誘導(dǎo)了細胞凋亡或增加了細胞膜通透性。Ndk對PA全基因組基因表達的影響。通過轉(zhuǎn)錄組測序檢測了PAO1及△ndk菌株基因表達差異。通過GO(gene ontology)功能注釋和KEGG(kyoto encyclopedia of genes and genomes)通路富集分析等生物信息學(xué)方法,考察Ndk對PA全基因組基因表達的影響。結(jié)果顯示,ndk基因敲除影響了PA物質(zhì)代謝/轉(zhuǎn)運等基礎(chǔ)生命活動相關(guān)基因表達。此外,ndk基因敲除影響了T3SS、胞外毒力因子合成等細菌毒力相關(guān)的基因表達。Ndk對PA毒力相關(guān)表型的影響。采用RT-q PCR、Western blot方法及相應(yīng)表型鑒定,檢測了PAO1、△ndk和△ndk+(ndk基因回補株)胞外毒力因子和T3SS相關(guān)的基因及蛋白表達情況、運動及生物膜形成能力。結(jié)果顯示,ndk基因敲除抑制了胞外毒力因子如:彈性蛋白酶、堿性磷酸酶、吩嗪等相關(guān)基因表達水平及彈性蛋白酶、綠膿素合成。然而,ndk基因敲除上調(diào)了T3SS基因及蛋白的表達水平。此外,ndk基因敲除抑制了PA集群運動及生物膜形成能力。Ndk對PA QS系統(tǒng)的調(diào)節(jié)作用。采用RT-q PCR、信號分子報告菌株平板法及高效液相色譜方法,檢測PAO1、△ndk和△ndk+菌株三大QS系統(tǒng)(las、rhl及pqs系統(tǒng))信號分子合成酶和轉(zhuǎn)錄因子相關(guān)基因的基因表達水平及信號分子產(chǎn)量。結(jié)果顯示,ndk基因敲除抑制了QS系統(tǒng)信號分子合成酶基因(las I、rhl I和pqs A)和轉(zhuǎn)錄因子基因(las R、rhl R和pqs R)的基因表達及信號分子(3-oxo-C12-HSL、C4-HSL和PQS)合成。Ndk通過QS系統(tǒng)調(diào)節(jié)PA毒力相關(guān)表型。通過在△ndk菌株培養(yǎng)基中添加外源性信號分子3-oxo-C12-HSL、C4-HSL或PQS,檢測△ndk菌株胞外毒力因子和T3SS相關(guān)基因的基因及蛋白表達水平。結(jié)果顯示,添加C4-HSL促進了△ndk菌株彈性蛋白酶及綠膿素的合成;而添加C4-HSL或PQS抑制了△ndk菌株上調(diào)的T3SS基因及蛋白表達水平。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R378
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本文編號：1379743