基于群体感应信号分子的抗菌剂对发光菌毒性的影响机制及预测模型

发布时间：2018-09-18 19:32

【摘要】：抗生素作为一种广谱性抗菌药物被广泛应用于医疗、畜牧业、养殖业等领域。然而,由于抗生素滥用及含抗生素废弃物的不合理处置,使环境中抗生素分布范围广度与环境范围浓度均日益提高。由于抗生素具有低剂量、持久性等一系列暴露特征,引发的抗性基因的产生给人类生存环境带来威胁,因而寻求抗生素替代品的需求日益迫切。随着抗生素替代品的提出及研究,如群体感应抑制剂(Quorum Sensing Inhibitor,QSIs),作为抗生素替代品被广泛研究,或与抗生素联合运用于医疗,可以预期在环境中,QSIs将与抗生素共存。然而,针对现阶段研究提出的群体感应抑制剂作为抗生素替代品的方案,其环境效应与环境生态风险影响等尚不明确。Vibrio fischeri作为一种生活在广阔海洋中的发光细菌,其产生的细菌群体感应信号分子C6(N-(3-oxo-hexanoyl)-L-Homoserine lactone)、C8(N-octanoyl-homoserine lactone)在环境中也存在相应分布,但是目前为止,对于环境中存在的C6、C8信号分子对抗菌剂作用于发光菌时的生物效应的影响的研究仍然很少。交叉现象是指根据单一污染物毒性试验数据用现有模型预测的剂量效应曲线与实验获得的污染物联合作用剂量效应曲线之间存在交点(一个或者多个)的现象,可为判别化合物之间的联合作用类型提供很好的依据,近年来被广泛研究。然而目前为止,交叉现象出现的机制尚不明确。本文以Vibrio fischeri为模式生物,磺胺类抗生素SMP、群体感应抑制剂类C30和群体感应信号分子C6、C8为研究对象,测定了C6、C8对抗生素和群体感应抑制剂单一和联合毒性效应(0-24h)及其基于IA模型的交叉现象的影响。研究发现,(1)信号分子C6对SMP、C30单一和联合作用于发光菌时的毒性效应有抑制作用,这是因为C6可与LuxR蛋白结合,启动luxICDABEG的表达,因而促进发光菌的发光。(2)低浓度的C8对SMP、C30单一和联合作用于发光菌时的毒性效应为抑制,高浓度的C8对SMP、C30单一和联合作用于发光菌时的毒性效应在0-12 h为抑制,13-24 h为促进,这是因为随着细菌自身分泌的C6增多,外源C8会跟C6竞争结合LuxR蛋白,使C6对发光菌的刺激作用减小。(3)C6会使SMP、C30联合作用于发光菌时出现交叉现象的浓度点增大,而C8使SMP、C30联合作用于发光菌时出现交叉现象的浓度点减小,这是因为C6、C8属于发光菌的两条不同的群体感应系统,C6属于LuxI/Lux R系统,C8属于AinS/AinR系统,在群体感应系统中C6比C8先发挥作用,而C8很大一部分作用是当C6浓度不足时,作为补充与LuxR蛋白结合,从而C6有使SMP+C30的联合毒性表现为拮抗的倾向,而C8有使SMP+C30联合毒性表现为协同的倾向。另外,面对当今污染物复杂多样、长时间暴露的严重情形,开展慢性毒性的研究就变得至关重要,然而目前对于急慢性毒性预测模型的研究大都为经验的选择,缺少基于急性与慢性毒性作用机制比较的理论依据,因此本文采用LitR蛋白的E_(binding)与Ka/Kc拟合的方法,建立了不同化合物同一个QSAR的从急性毒性预测慢性毒性的模型。研究发现,Lit R与靶蛋白之间有很好的相关性,这使得运用LitR的E_(binding)代替靶蛋白的E_(binding)与logKc/Ka进行拟合有了可行性。本文的研究结果不仅为抗菌剂联合作用的生态毒理评估和环境风险评价提供理论依据,而且还为交叉现象形成机制的进一步探索提供数据支撑和理论指导。
[Abstract]:As a broad-spectrum antibiotic, antibiotics are widely used in medical, animal husbandry, aquaculture and other fields. However, due to the abuse of antibiotics and unreasonable disposal of antibiotic-containing wastes, the distribution and concentration of antibiotics in the environment are increasing day by day. With the development of antibiotic substitutes, such as Quorum Sensing Inhibitor (QSIs), as an antibiotic substitute, it is widely studied or used in combination with antibiotics in medicine. It is expected that QSIs will coexist with antibiotics in the environment. However, the environmental effects and environmental ecological risk effects of quorum sensing inhibitors as alternatives to antibiotics are still unclear. Vibrio fischeri, a luminescent bacterium living in a vast ocean, produces a sense of bacterial community. The signal molecules C6 (N-(3-oxo-hexanoyl) -L-Homoserine lactone) and C8 (N-octanoyl-homoserine lactone) are also distributed in the environment, but so far, the effects of C6 and C8 signaling molecules on the biological effects of antimicrobial agents acting on luminescent bacteria in the environment are seldom studied. There is a phenomenon of intersection (one or more) between the dose-effect curves predicted by existing models and the dose-effect curves obtained by experiments, which can provide a good basis for identifying the types of combined action between compounds. However, the phenomenon of intersection has been widely studied in recent years. In this study, the effects of C6 and C8 on the single and combined toxicity (0-24 h) of antibiotics and quorum sensing inhibitors and their cross-phenomena based on IA model were investigated using Vibrio fischeri as model organism, sulfonamide antibiotics SMP, quorum sensing inhibitors C30 and quorum sensing signal molecules C6, C8. It was found that (1) signal molecule C6 inhibited the toxic effect of SMP and C30 on luminescent bacteria, because C6 could bind to LuxR protein and activate the expression of luxICDABEG, thus promoting the luminescence of luminescent bacteria. (2) The toxic effect of low concentration C 8 on SMP and C30 on luminescent bacteria was inhibited, and the toxic effect of high concentration C on luminescent bacteria was inhibited. The toxic effect of C8 on SMP, C30 on luminescent bacteria was inhibited at 0-12 h and promoted at 13-24 h, because exogenous C8 would compete with C6 to bind LuxR protein, and the stimulating effect of C6 on luminescent bacteria would decrease with the increase of C6 secreted by the bacteria themselves. (3) C6 would make the concentration point of cross-reaction between SMP and C30 on luminescent bacteria. C8 increased the concentration of SMP and C30, but decreased the concentration of C6 and C8, because C6 and C8 belonged to two different quorum sensing systems of luminescent bacteria. C6 belonged to LuxI/Lux R system, C8 belonged to AinS/AinR system, C6 played a role first than C8 in quorum sensing system, and C8 played a major role when the concentration of C6 was insufficient. As a complement to LuxR, C6 tends to antagonize the combined toxicity of SMP + C30, whereas C8 tends to synergize the combined toxicity of SMP + C30. In addition, chronic toxicity research is crucial in the face of the complex and diverse contaminants and the serious situation of long-term exposure. Sexual toxicity prediction models are mostly empirical choices, and lack of theoretical basis for comparing the mechanisms of acute and chronic toxicity. In this paper, the E_ (binding) and Ka/Kc fitting methods of LitR protein were used to establish a model for predicting chronic toxicity from acute toxicity of different compounds with the same QSAR. There is a good correlation between white and white, which makes it possible to use LitR E_ (binding) instead of target protein E_ (binding) to fit logKc / Ka. The results of this study not only provide a theoretical basis for ecotoxicological assessment and environmental risk assessment of antimicrobial agents, but also provide a further exploration for the formation mechanism of cross phenomena. Provide data support and theoretical guidance.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X171.5

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