铜绿假单胞菌胁迫条件下持久表达基因的研究

发布时间：2018-08-12 13:55

【摘要】： 铜绿假单胞菌(Pseudomonas aeruginasa,PA)是一种革兰氏阴性细菌,是医院感染的三大致病菌之一,能够引起致命性的急慢性感染。铜绿假单胞菌具有多重耐药机制,并能在贫营养、重污染等条件下抵抗胁迫生存,持久存活于肺纤维囊肿(Cystic Fibrosis,CF)病人体内。在治疗铜绿假单胞菌感染的过程中,有少量菌体在胁迫条件下存活于人体内,在一定的条件下再次引起感染,使得慢性病人长期反复感染,难以治愈。因此研究PA的持久生存机制,在此基础上开发可以有效阻断与病原菌持久存活相关的路径,对于有效控制临床上铜绿假单胞菌的长期感染,打破传统抗生素易于产生耐药性的局限性具有重要意义。本研究对以luxCDABE为报道子的铜绿假单胞菌PAO1随机启动子库(约3456个克隆)进行筛选,寻找能够在胁迫条件下引起持久发光的启动子,通过测序、比对,确定这些启动子相应的基因位置。挑选出其中具有长期条件下表达典型的基因进行敲除。对突变体与野生型菌株进行比较:在胁迫条件下长期培养并通过菌落形成单位(CFU)测其各自的存活率;对野生型与突变株进行共培养,让其在竞争状态下长期培养,然后分别测定其存活率;设计长时间培养的高渗透与低pH的胁迫条件来测定突变株在外加胁迫条件下的适应性。实验结果表明,从铜绿假单胞菌PAO1随机启动子库中筛选出157株在胁迫条件下长期表达的菌株,其启动子对应的基因被假定与PAO胁迫条件下生存相关。通过基因比对,最后确定了45个与持久生存相关的基因,其中14个是推测生物合成代谢相关基因,5个是假定的编码转录调节子,4个是假定的转运蛋白,另有4个已知基因和18个功能完全未知的基因。挑选其中7个(PA0423、PA1216、PA2827、PA0550、PA0256、PA0057、PA4578)重复性高且在在持久存活过程中表达明显的基因作为实验的目的基因重点研究,利用基因敲除技术分别使目的基因功能缺失,得到7株突变株。通过对比实验,结果表明:这七个基因均与菌体持久生长相关,其中五个基因(PA1216,PA0423,PA2827,PA0057,PA4578)在持久生存中起到重要作用,这些基因功能的缺失,造成细菌存活率明显下降。本研究结果初步阐明了这七个相关基因和铜绿假单胞菌在胁迫条件下持久存活的关系,但其作用机理还需进一步深入研究。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas) is a gram-negative bacteria, one of the three major nosocomial infections, which can cause fatal acute and chronic infections. Pseudomonas aeruginosa has the mechanism of multidrug resistance, and can survive under the condition of poor nutrition and heavy pollution, and can survive in patients with Cystic Fibrosis CF for a long time. In the course of treatment of Pseudomonas aeruginosa infection, a small number of bacteria survived in the human body under stress, and caused the infection again under certain conditions, which made chronic patients repeatedly infected for a long time and difficult to cure. Therefore, by studying the persistent survival mechanism of PA, we can effectively block the pathway related to the persistent survival of pathogenic bacteria and control the long-term infection of Pseudomonas aeruginosa clinically. It is of great significance to break the limitation that traditional antibiotics are prone to drug resistance. In this study, the random promoter library of Pseudomonas aeruginosa PAO1 (about 3456 clones) was screened with luxCDABE as reporter. The corresponding gene location of these promoters was determined. The genes with long term expression were selected for knockout. The mutants were compared with wild-type strains. The survival rate was measured by colony forming unit (CFU). The wild type and mutant were co-cultured for a long time under competitive condition. Then the survival rate of the mutant was determined, and the adaptability of the mutant under external stress was determined by designing the stress conditions of high osmotic and low pH for a long time. The results showed that 157 strains of Pseudomonas aeruginosa PAO1 promoter library were screened for long term expression under stress, and the corresponding gene of the promoter was assumed to be related to the survival of Pseudomonas aeruginosa under PAO stress. Through gene alignment, 45 genes related to persistent survival were identified, of which 14 were conjectural biosynthesis related genes, 5 were assumed transcriptional regulators, and 4 were assumed transporters. There were also 4 known genes and 18 completely unknown genes. Seven of them (PA0423, PA1216, PA2827, PA0550, PA0256, PA0057, PA4578) were selected as the key genes of the experiment. Seven mutants were obtained by using gene knockout technique. The results of comparative experiments showed that the seven genes were related to the sustained growth of bacteria, and five of them (PA1216 / PA0423 / PA2827PA0057 / PA4578) played an important role in the survival of the bacteria. The absence of these genes resulted in a significant decrease in the survival rate of bacteria. The relationship between the seven genes and the survival of Pseudomonas aeruginosa under stress was preliminarily clarified, but the mechanism of these seven genes should be further studied.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

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