纳米氧化铝促进多重耐药质粒RP4接合转移及其机理探讨
发布时间：2018-10-22 10:36
【摘要】： 可自主转移质粒在环境中的接合转移对细菌耐药性的传播起着重要作用。耐药细菌可通过食物链进入人体,从而加剧细菌耐药性问题。纳米材料和良好的应用前景,其广泛使用必然会导致水环境中的纳米材料增加。因其独特的物理化学特性,如小尺寸和大比表面积等,纳米材料具有很高的反应活性,可能会影响质粒的接合转移,目前还没有相关报道。本工作以携带多重耐药接合型质粒RP4的E.coli HB101为供体菌,Salmonella aberdeen Kauffmann 50312为受体菌,研究了纳米氧化铝对液相接合条件下RP4接合转移的影响及影响规律,采用形态学、生物化学、分子生物学以及蛋白质组学的方法和手段,探讨纳米氧化铝对RP4接合转移的影响机制。 本工作首先探讨了供、受体菌菌液浓度约为109cfu/mL(浓度比为1:3),接合8小时后,0.005~5mmol/L纳米氧化铝对转接合子数的影响。结果表明RP4的接合转移随着纳米Al2O3浓度的升高具有上升趋势,5mmol/L和50mmol/L纳米Al2O3组接合率分别为空白对照组的150倍和40倍,与空白对照组相比具有非常显著的差异。TEM的观察结果显示接合细菌的细胞外膜互相融合形成致密电子带。空白对照组虽然能观察到许多细菌紧密接触,但只有单个供、受体菌形成接合连接,5mmol/L和50mmol/L纳米Al2O3组能观察到多个细菌同时发生接合。 第二部份探讨了纳米氧化铝对接合转移的影响与接合时间、接合细菌的初始浓度、温度和pH值的关系。接合菌浓度105cfu/ml时,无论是否有纳米氧化铝干预,转接合子数始终随时间的延长而增加,0.5mmol/L Al2O3能缩短转接合子出现的时间,并且在接合时间内(90h)转接合子数增加的频率始终非常显著高于空白对照组,而5mmol/L和50mmol/L纳米氧化铝始终抑制接合。接合菌液浓度升高至106~108cfu/ml,接合8h时,纳米材料对接合的影响与初始接合菌液和纳米材料的浓度均相关。当接合菌浓度为108cfu/ml时,50mmol/L Al2O3能非常显著地提高转接合子数(P0.001),其它菌液浓度时对接合无影响;5mmol/LAl2O3始终非常显著地促进RP4接合转移,转接合子数分别为相应空白对照组的100~400倍(P0.001),在所有的组中促进作用最为显著;0.5mmol/L Al2O3能显著促进接合液浓度为106cfu/mL和107cfu/ml时的RP4的接合转移(P0.05)。温度(15℃、20℃、25℃、30℃、35℃)和pH值(6.0, 6.5, 7.0, 7.5, 8.0)对接合转移率无显著影响。 为研究纳米氧化铝对RP4接合的促进作用与其浓度之间的关系,采用激光扫描共聚焦显微镜(lazer scanning confocal microscope,LSCM)和TEM观察纳米氧化铝在细菌中的分布情况及对细菌超微结构的影响,并检测细菌抗氧化系统的变化情况。LSCM观察结果表明50mmol/L纳米Al2O3组荧光强度高于5mmol/L纳米Al2O3组。TEM观察结果显示纳米氧化铝能在细菌胞内沉积,5mmol/L纳米Al2O3组细菌胞质凝聚,而50mmol/L Al2O3组部份细菌观察不到完整的细胞壁和细胞膜结构。检测0.005~5mmol/L纳米氧化铝干预对细菌抗氧化酶系统的影响,结果表明5mmol/L和50mmol/L Al2O3干预后,细菌的总抗氧化能力(T-AOC)升高,相应的抗氧化酶如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)以及谷胱苷肽还原酶(GR)活力均显著高于空白对照组,其它组则无显著差异。 IncP质粒接合转移需要接合基因的参与,但是接合基因并非组成型表达。纳米氧化铝可能会影响接合基因的表达。采用启动子融合技术,构建trbBp-lacZ和trfAp-lacZ转录融合子,观察纳米Al2O3干预对接合配接对形成系统(mating pair formation,Mpf)和DNA转移及复制系统(DNA transfer and replication,Dtr)转录的影响。结果表明5mmol/L和50mmol/L纳米Al2O3组trbBp和trfAp的β-半乳糖苷酶活力显著高于空白对照组,5mmol/L和50mmol/L纳米Al2O3组之间β-半乳糖苷酶活力无显著差异。 接合基因的表达受整体调节因子调控,应用实时荧光定量PCR的方法检测整体调控因子korA、korB和trbA mRNA的表达水平,观察0.05~5mmol/L纳米Al2O3干预对整体调控因子korA、korB和trbA mRNA表达的影响,结果显示与空白对照组相比,5mmol/L和50mmol/L纳米氧化铝作用后korB和trbA的表达量均显著上升,korA mRNA的表达水平与空白对照组具有上升趋势,但是没有显著差异。5mmol/L和50mmol/L纳米氧化铝间各基因的mRNA表达量也没有显著差异。 采用二维聚丙烯酰胺凝胶电泳(2-D PAGE)及质谱技术,分析5mmol/L纳米氧化铝干预对蛋白质谱表达的影响,从蛋白水平探讨响应纳米氧化铝效应的蛋白。根据2D-PAGE结构,选取其中6个在5mmol/L纳米氧化铝组表达上调的蛋白进行质谱分析,成功地鉴定了3个蛋白。其中2个蛋白为供体菌E.coli HB101表达,分别为苹果酸合酶G和丙酮酸激酶,另一个蛋白为受体菌MS1表达,是一种可能的过氧化物酶。 上述结果表明: 1.纳米Al2O3能提高RP4介导的接合转移,可能从而提高水环境中细菌的耐药水平。纳米Al2O3对转接合子数的影响水平与纳米Al2O3及接合菌液的浓度有关。随着接合菌液浓度降低,能最大地促进RP4接合转移的纳米Al2O3的浓度也降低。 2.纳米氧化铝能够诱导产生活性氧自由基(reactive oxygen species ,ROS),损伤细菌的细胞膜和细胞壁结构。氧化铝对细菌细胞膜结构的轻微损伤作用可能有利于接合的发生,但是当细胞膜严重损伤时则不利于接合进行。 3.纳米氧化铝能促进整体调节因子korB和trbA的转录,以调控供体菌和受体菌中trfAp和trbBp的表达,使接合液中Dtr系统和Mpf系统的基因转录水平均升高,从而促进RP4接合转移。 4.纳米氧化铝干预后RP4接合转移增加,该过程需启动许多基因表达,消耗大量能量,因此促进供体菌中能量代谢相关的酶表达升高。受体菌中产生一种未知的过氧化物酶,该酶可能参与了响应纳米氧化铝的氧化胁迫效应。 本工作首次提出纳米氧化铝具有促进水环境中耐药质粒接合转移的现象,并且初步探讨了其机理,提出了纳米材料可能会促进细菌耐药性在水环境中的传播,危害水环境安全,并有可能促进社区获得性病原菌的耐药性。该研究丰富了纳米材料对健康风险理论,为纳米材料的污染防护理论提供了理论依据。
[Abstract]:The transfer of autonomously transferable plasmids in the environment plays an important role in the propagation of bacterial resistance. The drug-resistant bacteria can enter the human body through the food chain, thus increasing the problem of bacterial resistance. Nanomaterials and good application prospects, their widespread use necessarily lead to an increase in nanomaterials in water environments. Due to its unique physical and chemical properties, such as small size and large specific surface area, nano-materials have very high reaction activity, which may affect the conjugation and transfer of plasmids, and there are no relevant reports at present. coli HB101 containing multiple drug-resistant binding plasmid RP4 was donor strain, Salmonella aberdeen Kaufmann 50312 was the acceptor strain, and the effect of nano-alumina on the transfer of RP4 was studied. Molecular biology and proteomics methods and methods were used to investigate the effect mechanism of nano-alumina on the transfer of RP4. At the beginning of this work, the concentration of bacteria liquid for donor and recipient is about 109cfu/ mL (concentration ratio is 1: 3), after 8 hours of conjugation, 0. 005-5mmol/ L nano-alumina is used to transfer the zygotes. The results showed that the bonding transfer of RP4 increased with the increase of the concentration of nano-Al2O3, and the bonding rate of 5mmol/ L and 50mmol/ L nano-Al2O3 groups was 150 times and 40 times of the blank control group, and it was very significant compared with the blank control group. The results of TEM showed that the outer membrane of the jointed bacteria fused with each other to form a dense layer. Although many bacteria were found to be in close contact with the blank control group, only a single donor was used to form a joint connection, and the 5mmol/ L and 50mmol/ L nano-Al2O3 groups were able to observe multiple bacteria at the same time The second part discusses the effect of nano-alumina on bond transfer and bonding time, the initial concentration and temperature of bonding bacteria. When the concentration of the bacteria was 105cfu/ ml, the number of zygotes increased with the prolongation of time, and 0.5mmol/ L Al2O3 could shorten the time when the zygotes appeared, and the frequency of the increase of the number of zygotes in the time of conjugation (90h) was always very significant. Above blank control group, 5mmol/ L and 50mmol/ L nano-oxidation Aluminum always inhibits bonding. When the concentration of jointed bacteria is raised to 106-108cfu/ ml, the effect of nano-materials on bonding is related to the initial bonding bacteria liquid and nano material at 8h. When the concentration of jointed bacteria was 108cfu/ ml, 50mmol/ L Al2O3 could significantly improve the number of zygotes (P0. 001) and the concentration of other bacteria liquid had no effect on the conjugation; 5mmol/ L _ 2O _ 3 always promoted the transfer of RP4, and the number of zygotes was 100 ~ 400 times higher than that of the corresponding blank control group (P <0.05). P0. 001), most significant in all groups; 0. 5mmol/ L Al2O3 significantly contributed to the junction transfer of RP4 when the concentration of the conjugate was 106cfu/ mL and 107cfu/ ml (P0.05). The temperature (15 鈩，
本文编号：2286919
[Abstract]:The transfer of autonomously transferable plasmids in the environment plays an important role in the propagation of bacterial resistance. The drug-resistant bacteria can enter the human body through the food chain, thus increasing the problem of bacterial resistance. Nanomaterials and good application prospects, their widespread use necessarily lead to an increase in nanomaterials in water environments. Due to its unique physical and chemical properties, such as small size and large specific surface area, nano-materials have very high reaction activity, which may affect the conjugation and transfer of plasmids, and there are no relevant reports at present. coli HB101 containing multiple drug-resistant binding plasmid RP4 was donor strain, Salmonella aberdeen Kaufmann 50312 was the acceptor strain, and the effect of nano-alumina on the transfer of RP4 was studied. Molecular biology and proteomics methods and methods were used to investigate the effect mechanism of nano-alumina on the transfer of RP4. At the beginning of this work, the concentration of bacteria liquid for donor and recipient is about 109cfu/ mL (concentration ratio is 1: 3), after 8 hours of conjugation, 0. 005-5mmol/ L nano-alumina is used to transfer the zygotes. The results showed that the bonding transfer of RP4 increased with the increase of the concentration of nano-Al2O3, and the bonding rate of 5mmol/ L and 50mmol/ L nano-Al2O3 groups was 150 times and 40 times of the blank control group, and it was very significant compared with the blank control group. The results of TEM showed that the outer membrane of the jointed bacteria fused with each other to form a dense layer. Although many bacteria were found to be in close contact with the blank control group, only a single donor was used to form a joint connection, and the 5mmol/ L and 50mmol/ L nano-Al2O3 groups were able to observe multiple bacteria at the same time The second part discusses the effect of nano-alumina on bond transfer and bonding time, the initial concentration and temperature of bonding bacteria. When the concentration of the bacteria was 105cfu/ ml, the number of zygotes increased with the prolongation of time, and 0.5mmol/ L Al2O3 could shorten the time when the zygotes appeared, and the frequency of the increase of the number of zygotes in the time of conjugation (90h) was always very significant. Above blank control group, 5mmol/ L and 50mmol/ L nano-oxidation Aluminum always inhibits bonding. When the concentration of jointed bacteria is raised to 106-108cfu/ ml, the effect of nano-materials on bonding is related to the initial bonding bacteria liquid and nano material at 8h. When the concentration of jointed bacteria was 108cfu/ ml, 50mmol/ L Al2O3 could significantly improve the number of zygotes (P0. 001) and the concentration of other bacteria liquid had no effect on the conjugation; 5mmol/ L _ 2O _ 3 always promoted the transfer of RP4, and the number of zygotes was 100 ~ 400 times higher than that of the corresponding blank control group (P <0.05). P0. 001), most significant in all groups; 0. 5mmol/ L Al2O3 significantly contributed to the junction transfer of RP4 when the concentration of the conjugate was 106cfu/ mL and 107cfu/ ml (P0.05). The temperature (15 鈩，
本文编号：2286919
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