【作者】 徐洋洋；

【导师】 段康民；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 近年来,随着抗生素的广泛应用,临床病原细菌的耐药性问题日益突出,己对人类健康构成严重威胁。铜绿假单胞菌(Pseudomonas aeruginosa, PA)作为一种革兰氏阴性机会致病菌,能够在临床中引发严重感染,是导致病人院内感染的第三大致病菌。由于其内在的高抗药性及获得性抗药性,由铜绿假单胞菌引发的感染己越来越难以治愈。研究铜绿假单胞菌的致病性和耐药机制,进而从新的角度防治铜绿假单胞菌引起的感染已变得极为重要。铜绿假单胞菌的致病性受信号传递系统的调控。本研究涉及的与铜绿假单胞菌致病性相关的调控通路包括铜绿假单胞菌的群体感应调节系统、低于抑制浓度抗生素信号分子及自诱导信号分子AI-2(Autoinducer-2)。铜绿假单胞菌的致病因子受菌种特异的(Species-specific)高丝氨酸内酯(Acyl-homoserine Lactone, AHL)介导的群体感应系统(Quorum Sensing Systems, QSS)和以喹诺酮(Quinolone)为信号分子的群体感应系统的调节。铜绿假单胞菌拥有两个基于AHL的系统,一个是Las系统,另一个是Rhl系统,而基于喹诺酮的群体感应系统的信号分子为PQS (Pseudomonas Quinolone Signal)和HHQ (2-heptyl-4-quinolone)。抗生素除了传统的杀菌或抑菌功能外,低于抑制浓度抗生素也可作为细菌间相互作用的信号分子,参与铜绿假单胞菌的基因调控。研究还表明,细菌通用(Universal)群体感应系统的信号分子A1-2可以调控铜绿假单胞菌的致病性。这些信号传递系统不仅能够调控铜绿假单胞菌的致病性,它们之间也存在着相互作用。细菌细胞间相互作用方面的研究不仅有利于了解病原菌的致病机理,而且也是以此为靶点开发新型药物的重要基础。本课题重点研究信号传递系统对铜绿假单胞菌致病性的调控机制以及信号传递系统之间的相互作用,其主要工作包括以下四个部分：(1)在低于抑制浓度抗生素对铜绿假单胞菌致病因子调控的研究中,由于前期研究表明,铜绿假单胞菌吩嗪化合物受低于抑制浓度四环素的调节。本课题在此基础上系统地研究了铜绿假单胞菌吩嗪合成受多种低于抑制浓度抗生素的调控作用。在基因组水平,利用饱和转座突变方法,筛选并研究了对吩嗪合成操纵子PhzA1B1C1D1E1F1G1(phzA1)和phzA2B2C2D2E2F2G2(phzA2)有调节作用的基因及其调控途径,尤其是与低于抑制浓度的壮观霉素调节相关的基因及调控途径。同时,以吩嗪合成操纵子为报道子,探索了群体感应调节系统与低于抑制浓度抗生素调节系统之间的关系。实验发现,低于抑制浓度的壮观霉素(Spectinomycin, Spc)可以激活phzA1和phzA2操纵子的表达。在加入壮观霉素的条件下,phzA1的表达比不加壮观霉素时高出30倍,phzA2的表达则提高了4倍。在低于抑制浓度壮观霉素的调节机理研究中,通过筛选约30,000个转座突变体,在铜绿假单胞菌PAO1的全基因组中寻找到了参与壮观霉素调节phzA1/phzA2表达的基因。其中lasI、mvfR、vqsM、pqsE属于正调节phzA1和phzA2表达的基因,与已知结果一致。实验同时还筛选得到8个基因,这8个基因的突变不同程度地影响了壮观霉素对phzA1/phzA2的调节,表明这些基因可能参与这一调节过程。其中,基因ppk和基因pcnB被破坏之后,壮观霉素对phzA1的表达比不加壮观霉素时有明显的升高,基因ppk编码多磷酸激酶,而pcnB编码poly(A)聚合酶。在以phzA2为报道子筛选的转座突变体中,基因pilH、 pilY和pilX的突变使壮观霉素对phzA2的激活增加,并且这些基因都与IV型菌毛合成蛋白有关。基因algC突变之后,壮观霉素对phzA2的激活降低,该基因编码磷酸甘露糖变位酶。基因truA和基因rsaL突变之后,壮观霉素对phzA2的激活作用消失,即在该突变体中,加入壮观霉素和不加的条件下,phzA2的表达没有变化,truA是tRNA假尿嘧啶合成酶I的编码基因,而rsaL编码的产物是铜绿假单胞菌内与QS系统有关的调节蛋白。(2)探究了铜绿假单胞菌群体感应调节系统对致病因子吩嗪合成操纵子调控的机制。现有研究表明,在铜绿假单胞菌PAO1的两个操纵子phzA1B1C1D1E1F1G1(phzA1)和phzA2B2C2D2E2F2G2(phzA2)中,大部分吩嗪合成是由操纵子phzA1负责完成。为进一步了解phzA1和phzA2在PAO1中的分工和不同调节系统对这两个操纵子作用的具体机制及其差别,本课题构建和收集了多个调节系统突变体,并通过实验在这些突变体中检验了两个操纵子表达的变化。结果表明在lasl/rhll和pqsR缺失的突变体中,phzAl和phzA2的表达均受到了抑制。而在双组分调控基因(Two-Component Regulatory System) gacA缺失的突变体中,phzA1的表达降低而phzA2的表达增强,从而表明gacA正调节phzA1而负调节phzA2。在△vqsR、ΔPA0011和ΔPA1243突变体中,phzA2受壮观霉素的调节增强,而phzA1受壮观霉素的调节作用不明显。上述结果揭示phzA1和phzA2调节系统的差异可能与这些基因相关。另外,在针对调节子LasR、RhlR以及PqsR与phzA1相互作用的研究中,发现调节蛋白LasR和RhlR并非直接结合在phzA1的启动子区域上的。(3)相关研究表明操纵子phzA1上游的启动子区域含有一个las-box元件,是群体感应调节系统的调控应答元件。而实验室前期研究表明低于抑制浓度的四环素对phzA1的调控也依赖于这一元件,含有las-box启动子的表达比不加las-box时的表达有所升高。因此,本课题证实了las-box与四环素的直接作用,las-box作为phzA1基因表达的活性位点,同时也是四环素直接调节phzA1表达的元件,本研究结果进一步证实了群体感应调节系统与低于抑制浓度抗生素调节系统之间的关系。(4)研究了信号分子AI.2对铜绿假单胞菌致病性的影响及其机理,结果表明加入外源的A1.2可对铜绿假单胞菌致病性产生重要影响。大鼠肺部感染动物模型实验结果显示,AI-2能够加重铜绿假单胞菌肺部感染对肺组织的破坏。与铜绿假单胞菌单独感染相比,AI-2的存在可使肺组织破坏程度加重。对AI-2作用机理的研究表明,AI-2能够调节铜绿假单胞菌中的Rhl系统,对基因rhlI、rhlR和rhlAB的表达有激活作用,同时还发现AI.2能够增加与Rh1系统相关的泳动能力。尽管A1-2能够激活Rh1系统中的主要基因,实验发现,当存在AI-2时,Rh1群体感应系统的信号分子C4-HSL对Rhl系统的激活作用降低。表明AI-2与Rhl系统中的信号分子C4-HSL具有竞争关系,从而使得AI-2存在的情况下,C4-HSL的活性降低。这些结果揭示AI.2对铜绿假单胞菌致病因子的调节很可能是通过Rhl群体感应系统实现的。更多还原

【Abstract】 In recent years, the antibiotic resistance of clinical pathogenic bacteria has become an increasingly severe problem as a result of the wide use of antibiotics. As a gram-negative bacteria and opportunistic pathogen, Pseudomonas aeruginosa is the third source of nosocomial infections and can cause serious infections during medical treatment. Additionally, the treatment of such infections is fairly difficult due to the high innate antibiotic and acquired resistance of P.aeruginosa. Thus, it is of significant meaning to carry out research into both the infection and innate antibiotic resistance mechanisms of P.aeruginosa, which will provide new measures for prevention and treatment of infections caused by P.aeruginosa.The pathogenesis of P. aeruginosa is modulated by signaling pathways. This study focuses on the following signaling pathways:quorum-sensing systems in P. aeruginosa, signal molecule of sub-inhibitory concentration antibiotics and inter-species signal autoinducer-2. All these three pathways are related to the modulation off P.aeruginosa pathogenesis. The virulence factors are modulated by species-specific acyl-homoserine lactone (AHL) mediated quorum sensing as well as quinolone mediated quorum sensing systems. There are two quorum-sensing systems based on the AHL signal, namely Las system and Rhl system. In addition, there is a third quorum sensing system based on the Pseudomonas quinolone signal (PQS) and2-heptyl-4-quinolone (HHQ). Besides the ability to inhibit the growth of and kill bacteria, antibiotics of sub-inhibitory concentrations can act as the signal between bacteria interaction and participate in gene regulation. Moreover, research also shows that signal AI-2between universal quorum-sensing systems can modulate the P. aeruginosa pathogenesis. Furthermore, these signaling pathways also interact with one another. Research of interaction between cells is of significance for understanding the pathogenesis mechanisms of pathogen, and it also provides a basis for the development of novel drugs.This thesis mainly focuses on the modulation mechanisms of signaling pathway on P. aeruginosa pathogenesis, as well as the interactions between these signaling pathways. The major work and contributions of this thesis are as follows:(1) This study investigates the phenazine regulation by sub-inhibitory concentration of antibiotics in P. aeruginosa. Based on existing research, this work reveals that phenazine is regulated by sub-inhibitory concentration of tetracycline. At the genome level, the transposon mutation method is used to screen out the genes and the regulation pathway, involved in regulation of operons phzA1B1C1D1E1F1G1(phzA1) and phzA2B2C2D2E2F2G2(phzA2), which are responsible for phenazine production, especially, the genes and pathways involved in regulation of phzAl and phzA2by spectinomycin. Meanwhile, the relationship between quorum-sensing system and sub-inhibitory antibiotic modulation is investigated based on operon phzA1/phzA2expression.In preliminary work, we have discovered that sub-inhibitory concentrations of spectinomycin can activate the expression of phzAl and phzA2. By adding spectinomycin, the expression of phzAl is30-fold higher and phzA2expression increases by4-fold. With the employment of the transposon mutation method, we screen the whole P.aeruginosa genome database for the genes involved in Spc mediated alternation of phzA1/phzA2expression. Among them, genes lasl, mvfR, vqsM,pqsE can positively regulate the expression of phzA1and phzA2, which well agrees with existing results. Besides, we have screened out eight genes that are identified to be related to the activation of spectinomycin for phzAllphzA2, indicating that these genes may be involved in this regulation. With the disruption of genes ppk and pcnB, the expression of phzA1increases after incubating with Spc. Ppk is a polyphosphate kinase while PcnB is a poly (A) polymerase. With the disruption of pilH, pilY and pilX, the expression of phzA2increases when exposed to Spc, indicating these three genes are related to the type4fimbrial biogensis protein production. In the algC deletion strain, activation of phzA2mediated by Spc was decreased, and algC encodes the phosphomannomutase. With the disruption of truA and rsaL, the alternation made by Spc disappeares, which indicates that Spc has no effect on phzA2expression in these two mutants. Thus, gene truA encodes the tRNA-pseudouridine synthase I while rsaL encodes the regulator protein related to quorum sensing in P.aeruginosa.(2) This work investigates the modulation mechanism on phenazine operons by quorum sensing systems in P. aeruginosa. Research shows that operons phzA1and phzA2in P. aeruginosa PAO1, which is opposite in PA14, most phenazine production is controlled by phzA1operon. In order to investigate the function of phzA1and phzA2, as well as the different regulation of these two genes in P. aeruginosa, we constructe and collecte several genes deletion strains which are the essential genes in regulatory systems in P. aeruginosa, and the promoter-reporter system of phzA1and phzA2are tested in the mutants. The study shows that, in the mutant△lasI/△rhll/△pqsR, the expression of phzA1and phzA2are inhibited. However, in the gacA deletion strain, the essential regulator in two-component regulatory system, the expression of phzAl was inhibited while the expression of phzA2was activated. The result above means gacA can positively regulate phzA1while negatively regulate phzA2. Additionally, in the mutants of vqsR, PA0011and PA1243, experiments demonstrate that the expression of phzA2mediated by Spc is activated more than control, while the activation of phzAl by Spc remaines unchanged. These phenomenons reveal the difference in phzAl and phzA2expression, represented by the distinct modulation of phzA1and phzA2, may be related to these genes. Meanwhile, in studying the regulation of LasR, RhlR and PqsR on phzA1, experimental results show that LasR and RhlR indirectly modulated phzA1instead of directly binding to phzA1promoter region.(3) While investigating the relationship between quorum-sensing system and antibiotics regulation at sub-inhibitory concentration, the element las-box, the modulation responding element, is found in the upstream region of operon phzA1promoter. Previous study has revealed that alternation of phzA1expression by sub-inhibitory concentration of tetracycline is dependent on las-box. The expression of phzA1is higher under the existence of las-box. Research work in this thesis directly confirms the interaction between las-box and tetracycline. Specifically, las-box is not the only activate element for phzA1expression, but also the element for tetracycline modulation of phzA1.(4) This thesis further studies the signal AI-2effection on pathogenesis of P. aeruginosa and its mechanisms. Study has found that exogenous AI-2can affect pathogenesis of P. aeruginosa. The rat lung model experiment demonstrates that the existing of AI-2can aggravate lung infection by P. aeruginosa. Compared with pure P.aeruginosa infected condition, the lung infection is heavier under the existence of AI-2. While investigating the mechanism of AI-2function, we find that AI-2can regulate the Rhl system by activating the expression of gene rhll, rhlR and rhlAB in P.aeruginosa. Furthermore, the swarming ability of P.aeruginosa can be activated by AI-2as well. Although, AI-2can activate the major genes in Rhl system, the study shows that when AI-2exists, the activity of signaling molecular C4-HSL decreases, which leads to the speculation that AI-2competes with C4-HSL in regulation. These results reveal that the modulation of P. aeruginosa pathogenesis by AI-2may be achieved through the regulation of Rhl quorum sensing system.更多还原