銅綠假單胞菌脅迫條件下持久表達基因的研究
發(fā)布時間：2018-08-12 13:55
【摘要】： 銅綠假單胞菌(Pseudomonas aeruginasa,PA)是一種革蘭氏陰性細菌,是醫(yī)院感染的三大致病菌之一,能夠引起致命性的急慢性感染。銅綠假單胞菌具有多重耐藥機制,并能在貧營養(yǎng)、重污染等條件下抵抗脅迫生存,持久存活于肺纖維囊腫(Cystic Fibrosis,CF)病人體內。在治療銅綠假單胞菌感染的過程中,有少量菌體在脅迫條件下存活于人體內,在一定的條件下再次引起感染,使得慢性病人長期反復感染,難以治愈。因此研究PA的持久生存機制,在此基礎上開發(fā)可以有效阻斷與病原菌持久存活相關的路徑,對于有效控制臨床上銅綠假單胞菌的長期感染,打破傳統(tǒng)抗生素易于產生耐藥性的局限性具有重要意義。 本研究對以luxCDABE為報道子的銅綠假單胞菌PAO1隨機啟動子庫(約3456個克隆)進行篩選,尋找能夠在脅迫條件下引起持久發(fā)光的啟動子,通過測序、比對,確定這些啟動子相應的基因位置。挑選出其中具有長期條件下表達典型的基因進行敲除。對突變體與野生型菌株進行比較:在脅迫條件下長期培養(yǎng)并通過菌落形成單位(CFU)測其各自的存活率;對野生型與突變株進行共培養(yǎng),讓其在競爭狀態(tài)下長期培養(yǎng),然后分別測定其存活率;設計長時間培養(yǎng)的高滲透與低pH的脅迫條件來測定突變株在外加脅迫條件下的適應性。 實驗結果表明,從銅綠假單胞菌PAO1隨機啟動子庫中篩選出157株在脅迫條件下長期表達的菌株,其啟動子對應的基因被假定與PAO脅迫條件下生存相關。通過基因比對,最后確定了45個與持久生存相關的基因,其中14個是推測生物合成代謝相關基因,5個是假定的編碼轉錄調節(jié)子,4個是假定的轉運蛋白,另有4個已知基因和18個功能完全未知的基因。挑選其中7個(PA0423、PA1216、PA2827、PA0550、PA0256、PA0057、PA4578)重復性高且在在持久存活過程中表達明顯的基因作為實驗的目的基因重點研究,利用基因敲除技術分別使目的基因功能缺失,得到7株突變株。通過對比實驗,結果表明:這七個基因均與菌體持久生長相關,其中五個基因(PA1216,PA0423,PA2827,PA0057,PA4578)在持久生存中起到重要作用,這些基因功能的缺失,造成細菌存活率明顯下降。本研究結果初步闡明了這七個相關基因和銅綠假單胞菌在脅迫條件下持久存活的關系,但其作用機理還需進一步深入研究。
[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.
【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R378
本文編號：2179263
[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.
【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R378
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