单核细胞增生李斯特菌超氧化物歧化酶在菌膜形成中的功能解析

发布时间：2018-06-02 04:05

本文选题：菌膜 + 单核细胞增生李斯特菌　；参考：《上海交通大学》2013年博士论文

【摘要】：单核细胞增生李斯特菌是一种常见的食源性致病菌，可以引起人类的李氏杆菌病。它在自然界中分布广泛，并可在食品加工设备上形成菌膜。菌膜是微生物粘附在介质表面形成的大量细菌群居的一种生存状态。菌膜状态下的单核细胞增生李斯特菌对消毒剂等逆境的抗性明显增强，因此会增加食品反复被污染的几率，对公共卫生和人类健康带来严重危害。揭示单核细胞增生李斯特菌的菌膜形成机制对防止和控制它的危害具有重要意义。本实验室前期构建了Tn917插入单核细胞增生李斯特菌（Listeriamonocytogenes4b G）基因组的突变子库，从中筛选到了菌膜形成能力增加的突变株LM-49，并确定了插入位点为lm.G_1771基因（编码一个假定的ABC转运子透性酶）。通过基因芯片和双向电泳技术，筛选野生型G和lm.G_1771基因缺失突变株（Δ1771）的差异表达基因和蛋白，发现超氧化物歧化酶（SOD）的表达量在Δ1771突变株中明显升高。本文在此基础上对单核细胞增生李斯特菌sod基因与lm.G_1771基因的相互关系及在菌膜形成中的作用进行了研究，并探究了抗氧胁迫相关基因与菌膜形成之间的关系，主要研究内容和结果如下： 1、同源重组载体的构建与突变株的筛选。（1）sod基因两端的同源臂连接于温敏型穿梭质粒pKSV7（含氯霉素抗性基因）中，成功构建了sod基因的同源重组载体pKSV7::sod14。（2）重组质粒分别电转化于感受态细胞G和Δ1771中，得到阳性转化子，经过双交换和抗性筛选得到sod基因单缺失突变株Δsod和lm.G_1771、sod双缺失突变株Δ1771Δsod，重组几率分别为0.46%（2/436）和0.38%（5/1300）。突变株的获得为深入研究SOD的功能以及与lm.G_1771的关系提供了必要的材料。 2、lm.G_1771基因与sod基因表达的相互影响。首先分别培养制备游离状态和菌膜状态的细胞，再利用RT-qPCR技术和酶谱分析技术测定野生型菌株G和Δ1771中的SOD表达量。结果显示，在游离状态下，Δ1771中的sod表达量与野生型相比没有发生明显变化；菌膜状态下，基因sod在Δ1771中的表达量提高了2.2倍，SOD的酶活增加了2.3倍。同时，菌膜状态下lm.G_1771基因在Δsod突变株与野生型菌株G中的表达量无明显差别。该结果表明，菌膜状态下lm.G_1771基因的缺失会使sod的表达量增加，但在游离状态下这种作用不明显；此外，菌膜状态下sod的缺失对lm.G_1771基因的表达无明显影响。 3、SOD在菌膜形成中的生理功能及其同lm.G_1771的关系。首先，利用96孔板结晶紫染色法和荧光显微镜对野生型菌株G和三株突变株（Δ1771，Δsod和Δ1771Δsod）的菌膜形成能力进行评定。其次，对这四株菌的生长能力、活性氧组分（ROS）产量和抗氧胁迫能力进行了比较。结果显示：（1）与Δ1771菌膜形成能力增强相反，Δsod和Δ1771Δsod的菌膜形成能力均明显下降，尤其是Δ1771Δsod减少地更明显；（2）Δ1771与野生型G的生长能力无明显区别，Δsod和Δ1771Δsod生长减慢并且培养相同时间形成的菌落明显偏小；（3）Δ1771与野生型G的ROS产量无明显差异，Δsod和Δ1771Δsod的ROS产量明显升高，，尤其是在Δ1771Δsod中升高的更多；（4）Δ1771对氧化剂甲基紫晶（methyl viologen, MV）的敏感性与野生型G相似，Δsod和Δ1771Δsod对MV表现出高度敏感性，并且Δ1771Δsod比Δsod对甲基紫晶的敏感性还要强。上述结果表明，SOD作为重要的抗氧胁迫酶在维持细胞生长中起到重要作用，它可以抑制细胞过量ROS的产生，对菌膜形成起到正调控作用。并且，SOD可以与lm.G_1771一起调控菌膜的形成，两者在抵御氧胁迫中具有协同作用。 4、氧化剂MV（ROS产生诱导剂）对菌膜形成的影响。选取5种血清型共10株单核细胞增生李斯特菌野生型菌株，分别对它们在TSB培养基和TSB+MV培养基中的菌膜形成能力进行比较。结果显示，1mM MV可以明显抑制菌膜的形成，并影响该菌在玻片上的聚集，表明ROS不利于菌膜的形成。该结果为SOD通过抑制ROS的产生来调控菌膜形成的这一假说提供了辅证。 5、双向电泳（2-DE）及质谱联用鉴定SOD调控的蛋白。运用PDQuest8.0软件对野生型和Δsod突变株的双向电泳结果进行分析，共找到差异表达2倍以上的蛋白14个，其中10个在Δsod中下调，4个在Δsod中上调。下调的蛋白涉及细胞调节子、代谢、生长和压力反应相关的酶类；上调的蛋白涉及代谢和细胞壁合成相关的酶类；并且部分差异蛋白在菌膜形成中起到重要作用。这些结果暗示了SOD在生长和抗性方面可能的作用方式，推测了SOD正调控菌膜形成的途径。 6、利用RT-qPCR技术分析抗氧胁迫相关基因在不同状态及不同突变株中的表达。研究涉及的抗氧胁迫相关基因共分为三类：直接参与抗氧胁迫的基因（kat和fri）；抗氧胁迫基因表达的反应调节子（perR和sigB）；氧化损伤DNA修复基因（recA）。结果显示：（1）游离状态下，Δ1771和Δsod中抗氧胁迫基因和反应调节子的表达均比野生型要低，表明lm.G_1771和sod对这些基因均有正调控作用，并且这两个基因不影响recA的表达；但在双突变株Δ1771Δsod中，recA基因的表达量明显升高，由于recA具有启动SOS来维持生存的功能，表明了sod基因和lm.G_1771基因在共同维持细胞的正常生长中起到重要作用。（2）菌膜的形成会导致部分抗氧胁迫基因的上调，尤其在sod缺失突变株中，表明了菌膜的形成有利于抗氧胁迫缺陷菌株抗氧能力的修复。（3）MV可以刺激机体产生过量的内源性ROS，在MV的作用下，所研究的目的基因在野生型G中均被诱导上调，表明菌膜中形成的氧胁迫适应机制比起单纯的氧化剂刺激引起的适应机制复杂得多。综上所述，ROS可以抑制菌膜的形成，菌膜的形成需要氧胁迫相关基因的参与。SOD可以消除机体代谢产生的过量ROS，抵御氧胁迫的损伤，影响抗性、代谢和生长相关酶类的表达，维持细胞的正常生长和菌膜的形成。并且，在菌膜形成中SOD的表达会受到lm.G_1771的影响，在抗氧胁迫中两者表现出协同效应。总之，SOD可与lm.G_1771基因编码的ABC转运子透性酶一起调控菌膜的形成，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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R378;R155

【相似文献】