本文選題：菌膜 + 單核細胞增生李斯特菌�。� 參考：《上海交通大學》2013年博士論文

【摘要】：單核細胞增生李斯特菌是一種常見的食源性致病菌，可以引起人類的李氏桿菌病。它在自然界中分布廣泛，并可在食品加工設備上形成菌膜。菌膜是微生物粘附在介質表面形成的大量細菌群居的一種生存狀態(tài)。菌膜狀態(tài)下的單核細胞增生李斯特菌對消毒劑等逆境的抗性明顯增強，因此會增加食品反復被污染的幾率，對公共衛(wèi)生和人類健康帶來嚴重危害。揭示單核細胞增生李斯特菌的菌膜形成機制對防止和控制它的危害具有重要意義。 本實驗室前期構建了Tn917插入單核細胞增生李斯特菌（Listeriamonocytogenes4b G）基因組的突變子庫，從中篩選到了菌膜形成能力增加的突變株LM-49，并確定了插入位點為lm.G_1771基因（編碼一個假定的ABC轉運子透性酶）。通過基因芯片和雙向電泳技術，篩選野生型G和lm.G_1771基因缺失突變株（Δ1771）的差異表達基因和蛋白，發(fā)現超氧化物歧化酶（SOD）的表達量在Δ1771突變株中明顯升高。本文在此基礎上對單核細胞增生李斯特菌sod基因與lm.G_1771基因的相互關系及在菌膜形成中的作用進行了研究，并探究了抗氧脅迫相關基因與菌膜形成之間的關系，主要研究內容和結果如下： 1、同源重組載體的構建與突變株的篩選。（1）sod基因兩端的同源臂連接于溫敏型穿梭質粒pKSV7（含氯霉素抗性基因）中，成功構建了sod基因的同源重組載體pKSV7::sod14。（2）重組質粒分別電轉化于感受態(tài)細胞G和Δ1771中，得到陽性轉化子，經過雙交換和抗性篩選得到sod基因單缺失突變株Δsod和lm.G_1771、sod雙缺失突變株Δ1771Δsod，重組幾率分別為0.46%（2/436）和0.38%（5/1300）。突變株的獲得為深入研究SOD的功能以及與lm.G_1771的關系提供了必要的材料。 2、lm.G_1771基因與sod基因表達的相互影響。首先分別培養(yǎng)制備游離狀態(tài)和菌膜狀態(tài)的細胞，再利用RT-qPCR技術和酶譜分析技術測定野生型菌株G和Δ1771中的SOD表達量。結果顯示，在游離狀態(tài)下，Δ1771中的sod表達量與野生型相比沒有發(fā)生明顯變化；菌膜狀態(tài)下，基因sod在Δ1771中的表達量提高了2.2倍，SOD的酶活增加了2.3倍。同時，菌膜狀態(tài)下lm.G_1771基因在Δsod突變株與野生型菌株G中的表達量無明顯差別。該結果表明，菌膜狀態(tài)下lm.G_1771基因的缺失會使sod的表達量增加，但在游離狀態(tài)下這種作用不明顯；此外，菌膜狀態(tài)下sod的缺失對lm.G_1771基因的表達無明顯影響。 3、SOD在菌膜形成中的生理功能及其同lm.G_1771的關系。首先，利用96孔板結晶紫染色法和熒光顯微鏡對野生型菌株G和三株突變株（Δ1771，Δsod和Δ1771Δsod）的菌膜形成能力進行評定。其次，對這四株菌的生長能力、活性氧組分（ROS）產量和抗氧脅迫能力進行了比較。結果顯示：（1）與Δ1771菌膜形成能力增強相反，Δsod和Δ1771Δsod的菌膜形成能力均明顯下降，尤其是Δ1771Δsod減少地更明顯；（2）Δ1771與野生型G的生長能力無明顯區(qū)別，Δsod和Δ1771Δsod生長減慢并且培養(yǎng)相同時間形成的菌落明顯偏�。唬�3）Δ1771與野生型G的ROS產量無明顯差異，Δsod和Δ1771Δsod的ROS產量明顯升高，，尤其是在Δ1771Δsod中升高的更多；（4）Δ1771對氧化劑甲基紫晶（methyl viologen, MV）的敏感性與野生型G相似，Δsod和Δ1771Δsod對MV表現出高度敏感性，并且Δ1771Δsod比Δsod對甲基紫晶的敏感性還要強。上述結果表明，SOD作為重要的抗氧脅迫酶在維持細胞生長中起到重要作用，它可以抑制細胞過量ROS的產生，對菌膜形成起到正調控作用。并且，SOD可以與lm.G_1771一起調控菌膜的形成，兩者在抵御氧脅迫中具有協同作用。 4、氧化劑MV（ROS產生誘導劑）對菌膜形成的影響。選取5種血清型共10株單核細胞增生李斯特菌野生型菌株，分別對它們在TSB培養(yǎng)基和TSB+MV培養(yǎng)基中的菌膜形成能力進行比較。結果顯示，1mM MV可以明顯抑制菌膜的形成，并影響該菌在玻片上的聚集，表明ROS不利于菌膜的形成。該結果為SOD通過抑制ROS的產生來調控菌膜形成的這一假說提供了輔證。 5、雙向電泳（2-DE）及質譜聯用鑒定SOD調控的蛋白。運用PDQuest8.0軟件對野生型和Δsod突變株的雙向電泳結果進行分析，共找到差異表達2倍以上的蛋白14個，其中10個在Δsod中下調，4個在Δsod中上調。下調的蛋白涉及細胞調節(jié)子、代謝、生長和壓力反應相關的酶類；上調的蛋白涉及代謝和細胞壁合成相關的酶類；并且部分差異蛋白在菌膜形成中起到重要作用。這些結果暗示了SOD在生長和抗性方面可能的作用方式，推測了SOD正調控菌膜形成的途徑。 6、利用RT-qPCR技術分析抗氧脅迫相關基因在不同狀態(tài)及不同突變株中的表達。研究涉及的抗氧脅迫相關基因共分為三類：直接參與抗氧脅迫的基因（kat和fri）；抗氧脅迫基因表達的反應調節(jié)子（perR和sigB）；氧化損傷DNA修復基因（recA）。結果顯示：（1）游離狀態(tài)下，Δ1771和Δsod中抗氧脅迫基因和反應調節(jié)子的表達均比野生型要低，表明lm.G_1771和sod對這些基因均有正調控作用，并且這兩個基因不影響recA的表達；但在雙突變株Δ1771Δsod中，recA基因的表達量明顯升高，由于recA具有啟動SOS來維持生存的功能，表明了sod基因和lm.G_1771基因在共同維持細胞的正常生長中起到重要作用。（2）菌膜的形成會導致部分抗氧脅迫基因的上調，尤其在sod缺失突變株中，表明了菌膜的形成有利于抗氧脅迫缺陷菌株抗氧能力的修復。（3）MV可以刺激機體產生過量的內源性ROS，在MV的作用下，所研究的目的基因在野生型G中均被誘導上調，表明菌膜中形成的氧脅迫適應機制比起單純的氧化劑刺激引起的適應機制復雜得多。 綜上所述，ROS可以抑制菌膜的形成，菌膜的形成需要氧脅迫相關基因的參與。SOD可以消除機體代謝產生的過量ROS，抵御氧脅迫的損傷，影響抗性、代謝和生長相關酶類的表達，維持細胞的正常生長和菌膜的形成。并且，在菌膜形成中SOD的表達會受到lm.G_1771的影響，在抗氧脅迫中兩者表現出協同效應�？傊琒OD可與lm.G_1771基因編碼的ABC轉運子透性酶一起調控菌膜的形成，在菌膜形成中起到重要作用。
[Abstract]:Lester bacteria monocytogenes, a common foodborne pathogen, can cause human listeriosis. It is widely distributed in nature and can form a membrane on food processing equipment. The membrane is a survival state of a large number of bacteria colonies by microorganisms on the surface of the medium. Mononuclear cells in the state of the membrane increase. The resistance of Lester bacteria to the adversities, such as disinfectants, is obviously enhanced, so it will increase the probability of repeated contamination of food and bring serious harm to public health and human health. It is of great significance to reveal the mechanism of the membrane formation of the monocytic accretion of the monocytic fungus to prevent and control its harm.
A mutant library of Tn917 (Listeriamonocytogenes4b G) genome was inserted into the genome of the Tn917 inserted mononuclear cell proliferation (Listeriamonocytogenes4b G) in our laboratory. The mutant strain LM-49 was screened for the increase of the membrane formation ability, and the insertion site was identified as the lm.G_1771 gene (encoding a hypothetical ABC transporter). The differentially expressed genes and proteins of the wild type G and lm.G_1771 gene deletion mutants (delta 1771) were screened, and the expression of superoxide dismutase (SOD) was significantly increased in the delta 1771 mutant strain. On this basis, the relationship between the SOD gene of Lester monocytic and the lm.G_1771 gene and the preparation of the lm.G_1771 gene in the formation of the membrane were studied on this basis. The relationship between the genes related to oxygen stress and the formation of biofilm was studied. The main contents and results were as follows:
1, construction of homologous recombinant vector and screening of mutant strains. (1) the homologous arm of the SOD gene is connected to the Wen Minxing shuttle plasmid pKSV7 (chloramphenicol resistant gene), and the homologous recombinant vector pKSV7: of the SOD gene is successfully constructed: sod14. (2) recombinant plasmid is converted to the receptor of G and delta respectively, and the positive transformants are obtained. SOD gene single deletion mutant delta SOD and lm.G_1771, SOD double deletion mutant delta 1771 delta SOD were obtained by exchange and resistance screening, and the recombination probability was 0.46% (2/436) and 0.38% (5/1300) respectively. The gain of the mutant strain provided the necessary material for the in-depth study of SOD's function and the relationship with lm.G_1771.
2, the interaction between the lm.G_1771 gene and the SOD gene expression. First, the cells were cultured to prepare the free state and the membrane state respectively. The SOD expression in the wild type strain G and the delta 1771 was measured by RT-qPCR technique and the enzyme spectrum analysis technique. The results showed that the expression of SOD in the delta 1771 was not obvious compared with the wild type in the free state. Under the condition of membrane, the expression of gene SOD in delta 1771 increased by 2.2 times, and the activity of SOD increased by 2.3 times. At the same time, there was no significant difference in the expression of lm.G_1771 gene between the delta SOD mutant and the wild type G under the membrane condition. The results showed that the deletion of lm.G_1771 gene in the membrane state would increase the expression of SOD, but in the course of swimming, the expression of SOD was increased. In addition, the absence of SOD in the membrane state had no significant effect on the expression of lm.G_1771 gene.
3, the physiological function of SOD in the formation of membrane and its relationship with lm.G_1771. First, the membrane formation ability of wild type strain G and three mutant strains (delta 1771, delta SOD and delta 1771 delta SOD) was evaluated by 96 pore crystal violet staining and fluorescence microscopy. Secondly, the growth ability of the four strains of bacteria, the yield of active oxygen component (ROS) and the anti oxygen threat were used. The results showed that: (1) the membrane formation ability of delta SOD and delta 1771 delta SOD decreased obviously in contrast to the enhancement of delta 1771 membrane formation ability, especially delta 1771 delta SOD, and (2) the growth ability of delta 1771 and wild type G had no obvious difference, delta SOD and delta 1771 delta SOD growth slowed down and cultivated the same time. (3) there was no significant difference in the ROS yield between Delta and wild type G, the ROS yield of delta SOD and delta 1771 delta SOD increased significantly, especially in the delta 1771 delta SOD; (4) the sensitivity of delta 1771 to the oxidant methyl (methyl viologen, MV) was similar to that of the wild type G, and the delta SOD and delta 1771 delta SOD were highly sensitive to those of the wild type G. Moreover, the sensitivity of delta 1771 delta sod is stronger than that of delta sod. The results show that SOD, as an important antioxidant enzyme, plays an important role in maintaining cell growth. It can inhibit the production of excess ROS and regulate the formation of the membrane. And SOD can regulate the formation of the membrane with lm.G_1771. It has synergistic effect in oxygen stress.
4, the influence of the oxidant MV (ROS inducer) on the formation of the membrane. A total of 5 serotypes of 10 wild strains of monocytic Lester bacteria were selected to compare the membrane formation ability of them in the TSB medium and the TSB+MV medium respectively. The results showed that 1mM MV could inhibit the formation of the membrane and influence the bacteria on the slide. Aggregation indicates that ROS is not conducive to the formation of biofilm. This result provides evidence for the hypothesis that SOD regulates biofilm formation by inhibiting ROS production.
5, two dimensional electrophoresis (2-DE) and mass spectrometry were used to identify the proteins regulated by SOD. Using PDQuest8.0 software, the two-dimensional electrophoresis results of wild and delta SOD mutant strains were analyzed, and 14 proteins more than 2 times of differential expression were found, of which 10 were downregulated in delta SOD and 4 in Delta sod. The down regulated proteins involved cell regulators, metabolism, growth and pressure. The up-regulated proteins involved metabolism and cell wall synthesis related enzymes; and some differential proteins play an important role in the formation of membrane. These results suggest the possible ways of SOD in growth and resistance, and speculate on the way SOD is regulating the formation of the membrane.
6, RT-qPCR technique was used to analyze the expression of anti oxygen related genes in different states and different mutant strains. The related genes involved in the study were divided into three categories: the genes directly involved in the anti oxygen stress (KAT and Fri), the reactive regulator of the oxygen stress gene expression (perR and sigB), and the oxidative damaged DNA repair gene (recA). (1) in free state, the expression of anti oxygen stress gene and reaction regulator in delta 1771 and delta SOD were lower than that of wild type, indicating that lm.G_1771 and SOD had positive regulation effect on these genes, and the two genes did not affect the expression of recA, but the expression of recA gene was significantly increased in the double mutant delta 1771 delta SOD, due to recA The function of activating SOS to maintain survival indicates that the SOD gene and lm.G_1771 gene play an important role in the common maintenance of normal growth of the cells. (2) the formation of the membrane may lead to the up-regulation of some anti oxygen stress genes, especially in the sod deletion mutant, indicating that the form of the membrane is beneficial to the oxygen resistance of the strain of the anti oxygen stress strain. (3) (3) it can stimulate the body to produce excessive endogenous ROS. Under the action of MV, the target genes are all up regulated in the wild type G, indicating that the adaptation mechanism of oxygen stress formed in the membrane is much more complicated than the adaptive mechanism caused by the pure oxidant stimulation.
To sum up, ROS can inhibit the formation of membrane. Membrane formation requires the involvement of oxygen stress related genes in the formation of.SOD, which can eliminate excessive ROS produced by the body metabolism, resist the damage of oxygen stress, influence resistance, metabolism and growth related enzymes, maintain the normal growth of cells and form the membrane of the cells, and the form of SOD in the formation of the membrane. It was affected by lm.G_1771 and showed synergistic effect in both oxygen resistance and stress. In conclusion, SOD can regulate the formation of the membrane with the ABC transporter of lm.G_1771 gene, which plays an important role in the formation of the membrane.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R378;R155

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