Fis介导伤寒沙门菌滞留菌形成的机制研究

发布时间：2018-11-15 16:33

【摘要】：目的:滞留菌是临床上慢性感染反复发作的重要原因之一,目前发现多种人类致病菌可形成滞留菌,滞留菌形成机制十分复杂,涉及细菌的毒力、能量代谢等环节。本研究将探讨伤寒沙门菌(Salmonella enterica serovar Typhi,S.Typhi)是否可形成滞留菌,环境因素对滞留菌形成的影响,伤寒沙门菌全局调控因子Fis在滞留菌形成中所发挥的作用及其调节机制。方法:1.药敏试验(纸片法):将麦氏浓度为0.5的菌液均匀涂布于M-H琼脂平板,氨苄西林(AMP)、环丙沙星(CIP)、卡那霉素(Kana)药敏纸片贴于平板表面,量取抑菌圈直径。2.最小抑菌浓度测定:于96孔培养板中将氨苄西林和环丙沙星倍比稀释至不同浓度,每孔中加入麦氏浓度为0.5的菌液100μL,24 h后能抑制细菌生长的最低药物浓度即为该菌株的最小抑菌浓度(MIC)。3.细菌药物存活曲线:伤寒沙门菌野生株培养至对数早期(OD600≈0.4),加入氨苄西林或环丙沙星作用,每隔1 h取100μL菌液稀释涂板计数。以药物作用时间为横轴,每个时间点菌落数为纵轴,绘制细菌在不同药物中的存活曲线。4.细菌滞留能力检测:将菌株培养至不同OD600值,加入氨苄西林或者环丙沙星作用5 h,取100μL菌液稀释后涂板计数。滞留率为5 h菌落数与未加入药物之前菌落数之比。5.q RT-PCR检测基因表达:伤寒沙门菌野生株、Δfis培养至一定的OD600值,TRIzol法提取细菌总RNA,逆转录后q RT-PCR检测,分析野生株中fis的表达情况,野生株和Δfis中谷氨酸运体相关基因,rel A和spo T的表达。6.RT-PCR验证glt操纵子结构:分别以glt I、glt L为上下游,以glt J、glt L为上下游各设计两对引物。伤寒沙门菌野生株培养至对数期(OD600≈0.4),提取细菌总RNA,用4个不同位置的下游引物逆转录。以获得的c DNA为模板,4对不同位置上下游引物进行PCR,琼脂糖凝胶电泳。7.伤寒沙门菌谷氨酸转运体和fis双缺陷株及回补株的构建:同源重组片段glt与自杀质粒p GMB151连接,阳性质粒电转至Δfis感受态构建缺陷株(记作ΔgltΔfis)。构建p BAD-glt重组质粒,阳性重组质粒和空质粒电转至转化ΔgltΔfis感受态,作为glt回补株(ΔgltΔfis+p BAD-glt)和空质粒对照株(ΔgltΔfis+p BAD)。结果:1.药敏试验结果表明,伤寒沙门菌野生株、fis基因缺失株对氨苄西林、环丙沙星、卡那霉素敏感。2.MIC测定结果说明,伤寒沙门菌野生株、fis基因缺失株对氨苄西林、环丙沙星敏感且两者对同一种药物敏感度相同。3.细菌药物存活曲线表明:氨苄西林、环丙沙星作用于伤寒沙门菌呈二项杀伤曲线变化,存在部分细菌稳定存活。4.伤寒沙门菌可形成滞留菌且滞留菌的产生与细菌的培养条件、生长时期相关。5.通过q RT-PCR分析:fis在对数早期表达量最高;相比野生株,在LB培养基中,Δfis的rel A、spo T表达量下降,而在特殊M9培养基中,相比野生株,Δfis的rel A、spo T表达量升高;Δfis中谷氨酸转运体相关基因gln H、gln P、glt I、glt K、glt J及glt S表达量升高相比野生株。6.相比野生株,fis基因缺失株滞留能力下降。7.相比普通LB培养基中的Δfis滞留率与野生株滞留率之间差异,特殊M9培养基中两者滞留率的差异缩小。8.成功构建伤寒沙门菌谷氨酸转运体和fis双缺陷株(ΔgltΔfis)及谷氨酸转运体回补株(ΔgltΔfis+p BAD-glt)。9.相比Δfis,ΔgltΔfis滞留能力升高;与空载体对照株相比,glt回补株(ΔgltΔfis+p BAD-glt)滞留能力降低。结论:伤寒沙门菌可形成滞留菌且受环境因素的影响,Fis介导伤寒沙门菌滞留菌的形成是通过调节谷氨酸转运体相关基因的表达,促进谷氨酸的转运,改变胞内的营养状态,从而影响滞留菌的形成,为进一步探索滞留菌形成的具体机制提供基础。
[Abstract]:Objective: The retention of bacteria is one of the important causes of the repeated episodes of chronic infection in the clinic. It is found that a variety of human pathogenic bacteria can form the detention bacteria, the mechanism of the formation of the detention bacteria is very complex, and it is related to the virulence and energy metabolism of the bacteria. The effect of Salmonella typhimurium (S. Typhi, S. Typhi) on the formation of retained bacteria, the effect of environmental factors on the formation of detention bacteria, the role of the global regulatory factor Fis in the formation of detention bacteria and its regulation mechanism will be discussed in this study. Method: 1. The drug sensitivity test (the paper-paper method): The bacterial liquid with the concentration of 0.5 was evenly coated on the surface of the plate, and the diameter of the bacteriostatic ring was taken as the diameter of the bacteriostatic ring. and the minimum inhibitory concentration (MIC) of the strain is the minimum inhibitory concentration (MIC) of the strain. The survival curve of the bacterial drug: the wild strain of Salmonella typhi was cultured to the logarithmic early stage (OD600-0.4), with the action of methicillin or ciprofloxacin, and 100. m u.L of bacterial liquid was used to dilute the coated plate at every 1 h. the time of the drug action is the horizontal axis, the number of the colonies per time point is the vertical axis, and the survival curves of the bacteria in different medicines are drawn. Test of the bacteria retention capacity: culture the strain to different OD600 values, add ampicillin or ciprofloxacin for 5 hours, and dilute the 100 & mu; L bacterial solution and then apply the plate to count. 5. q RT-PCR was used to detect the gene expression: the wild strain of Salmonella typhimurium, the yeast fids was cultured to a certain OD600 value, the total RNA of bacteria was extracted by the TRIzol method, and the expression of fids in the wild strain was analyzed by the reverse transcription q RT-PCR. The expression of glutamate transporter-related genes, rel A and spo T in wild plants and plants was verified by RT-PCR: glt I and glt L were the upstream and downstream of glt I and glt L, and two pairs of primers were designed for upstream and downstream of glt J and glt L. The wild strain of Salmonella typhi was cultured to the logarithmic phase (OD600-0.4), and the total RNA of the bacteria was extracted and reverse transcribed with the downstream primers at four different positions. and using the obtained c DNA as a template, 4 pairs of upstream and downstream primers are subjected to PCR and agarose gel electrophoresis. The construction of the glutamate transporter and the fids double-defective strain of the Salmonella typhi and the repair strain: the homologous recombination fragment glt is connected with the suicide plasmid pGMB151, and the positive plasmid is electrically transferred to the disulfide competent state to construct the defective strain (to be recorded as the yeast glt-fids). The pBAD-glt recombinant plasmid was constructed, and the positive recombinant plasmid and the empty plasmid were transferred to the transformed glt-fids competent state as the glt tfids + p BAD-glt and the empty plasmid control strain (Bglt-fids + p BAD). Results: 1. The results of drug sensitivity test showed that the wild strain of Salmonella typhi and the deletion of fids were sensitive to methicillin, ciprofloxacin and kanamycin. Ciprofloxacin is sensitive and both have the same sensitivity to the same drug. The survival curves of the bacterial drug show that the effect of methicillin and ciprofloxacin on Salmonella typhi is the change of two anti-kill curves, and some of the bacteria live stably. Salmonella typhi can form the detention bacteria and the generation of the detention bacteria is related to the culture conditions and the growth period of the bacteria. Through q-RT-PCR analysis, the expression of fids in the logarithmic early stage was the highest; in the LB medium, the expression of rel A and spo T of the fids decreased, while in the medium of the special M9, the expression of rel A and the spo T of the wild strain and FFIs increased. The expression of gln H, gln P, glt I, glt K, glt J and glt S in Glutamic acid transporter was higher than that of wild strain. Compared with the wild strain, the retention capacity of the fids gene is reduced. The difference between the retention rate and the retention rate of the wild strain in the medium of the normal LB medium was compared with that of the wild strain, and the difference of the retention rate of the two in the M9 medium was reduced. Salmonella typhimurium glutamate transporter and fids double-defective strain (tglt-fids + p BAD-glt) were successfully constructed. The retention capacity of glt-fids + p BAD-glt decreased compared with the empty vector control. Conclusion: Salmonella typhimurium can form the detention bacteria and is affected by the environmental factors, and the formation of the Fis-mediated Salmonella typhi is by regulating the expression of the related gene of the glutamate transporter, promoting the transport of the glutamic acid, changing the nutrient state in the cell, and thus affecting the formation of the detention bacteria, and provides the basis for further exploring the specific mechanism for the formation of the detention bacteria.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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