RpoN对鼠伤寒沙门菌生物被膜形成的调控作用研究
发布时间：2018-12-13 21:10
【摘要】：沙门菌可感染多种宿主,导致败血症、胃肠炎和腹泻,也可引起人的食物中毒,因此沙门菌病是影响全球公共卫生和经济发展最重要的问题之一。自然环境中,沙门菌以浮游状态和多细胞聚集的菌落如生物被膜状态存在。沙门菌通过附着在生物或非生物的表面形成生物被膜,抵抗消毒剂、环境压力、抗生素和宿主免疫系统的识别,从而促进细菌生存和传播,增强其毒力,与沙门菌病的爆发和患者持续感染相关。为了更好地防控人和动物的沙门菌病,有必要阐明沙门菌生物被膜形成的调控机制。本研究对两株生物被膜形成能力较强的鼠伤寒沙门菌利用Red同源重组系统构建rpoN基因缺失株和回复株,测定其生物被膜形成能力及成分的表达差异,发现RpoN为鼠伤寒沙门菌生物被膜形成相关σ因子之一;运用实时荧光定量PCR的方法测定鼠伤寒沙门菌野生株和rpoN基因缺失株在生物被膜形成过程中下游调控基因的转录水平差异,对转录水平发生明显上调的基因在野生株和rpoN基因缺失株的基础上分别构建缺失株,测定其生物被膜形成能力,进一步确定rpoN基因在鼠伤寒沙门菌生物被膜形成过程中的调控网络。1.鼠伤寒沙门菌rpoN基因缺失株的构建及生物学特性研究对两株生物被膜形成能力较强的鼠伤寒沙门菌利用Red同源重组系统构建rpoN基因缺失株,利用原核表达载体构建回复株;比较野生株、缺失株和回复株的生物被膜形成能力和对环境应激的抵抗力差异,并通过建立的csgA和bcsA基因实时荧光定量PCR方法检测了其生物被膜成分的表达差异。结果显示,与野生株相比,rpoN基因缺失株生物被膜形成能力增强,主要是卷曲菌毛蛋白表达量显著增加引起的。回复株生物被膜形成能力与野生株相似。rpoN基因缺失株在酸性应激和碱性应激条件下对外界环境应激的抵抗力均显著增强,说明rpoN基因为鼠伤寒沙门菌中生物被膜形成相关σ因子之一,这一发现有助于进一步研究沙门菌生物被膜形成的调控机制。2.RpoN在鼠伤寒沙门菌生物被膜形成过程中对下游基因的调控作用以gyrB基因为内参,运用实时荧光定量PCR的方法测定鼠伤寒沙门菌野生株和rpoN基因缺失株在生物被膜形成过程中的下游调控基因csgD、csgA、bcsA、adrA、gcpA、fimA、lpfE、hfq的转录水平差异,发现rpoN基因缺失后,csgD、csgA基因在生物被膜状态下4 h、8h和24h这3个时间段转录水平均呈现极显著增强(P0.01),gcpA、fimA基因在8h转录水平极显著提高(P0.01),lpfE、hfq基因也在8h转录水平有显著增强(P0.05),bcsA、adrA基因转录水平却没有显著变化。在野生株和rpoN基因缺失株的基础上分别构建adrA、gcpA、fimA、lpfE基因缺失株,测定其生物被膜形成能力。结果显示,与野生株相比,adrA基因单缺失株的生物被膜形成能力显著下降(P0.05),而gcpA、fimA、lpfE基因缺失后对其生物被膜形成能力没有明显的影响;与rpoN基因单缺失相比,在其基础上缺失adrA基因得到的双缺失株生物被膜形成能力显著下降(P0.05),缺失gcpA基因得到的双缺失株生物被膜形成能力极显著下降(P0.01),与野生株相似。缺失fimA或fpfE基因得到的双缺失株生物被膜形成能力没有明显的影响。通过实时荧光定量PCR测定两株鼠伤寒沙门菌rpoN基因单缺失株中rpoS基因的表达量以及rpoS基因单缺失株中rpoN基因的表达量,与野生株相比,rpoN基因和rpoS基因的表达量均没有显著差异,即在鼠伤寒沙门菌生物被膜形成过程中σ因子RpoN与RpoS之间没有明显的相关性。因此,rpoN基因可能通过负调控gcpA基因增强鼠伤寒沙门菌生物被膜的形成。
[Abstract]:Salmonella can infect a variety of hosts, resulting in sepsis, gastroenteritis and diarrhea, and can also cause food poisoning in humans, so Salmonella is one of the most important issues affecting global public health and economic development. In the natural environment, Salmonella are present in a suspended state and in a multicell-aggregated colony, such as a biological membrane. The salmonella forms a biological membrane by attachment to the surface of a biological or non-biological surface to resist the identification of the disinfectant, the ambient pressure, the antibiotic and the host immune system, thereby promoting the survival and transmission of bacteria, enhancing its virulence, and related to the outbreak of Salmonella and the continuous infection of the patient. In order to prevent and control the salmonellosis of human and animal, it is necessary to clarify the regulation mechanism of the formation of the biological membrane of Salmonella. By using the Red homologous recombination system to construct the rpoN gene deletion strain and the restoring strain, the strain of Salmonella typhimurium which has stronger ability to form two biofilms is determined by using the Red homologous recombination system, and the expression difference of the biological membrane forming ability and the component is determined, and the RpoN is found to be one of the factors related to the formation of the membrane of the Salmonella typhimurium. in that method, a real-time fluorescence quantitative PCR method is used to measure the difference of the transcription level of the wild strain and the rpoN gene deletion strain of the mouse typhi in the middle and lower reaches of the biological membrane-forming process, On the basis of the deletion of the wild strain and the rpoN gene, a deletion strain was constructed on the basis of the deletion of the wild strain and the rpoN gene, and the ability of its biological membrane formation was determined to further determine the regulation and control network of rpoN gene in the process of membrane formation of Salmonella typhimurium. the construction and biological characteristics of the rpoN gene deletion strain of the Salmonella typhimurium are used to construct the rpoN gene deletion strain by using the Red homologous recombination system to construct the rpoN gene deletion strain by using the Red homologous recombination system, The biological membrane-forming ability and the resistance to environmental stress of the deleted strain and the restoring strain are different, and the expression difference of the biological membrane components is detected by the established csgA and bcsA gene real-time fluorescence quantitative PCR method. The results showed that the loss of rpoN gene was enhanced by the membrane-forming ability as compared with the wild strain, which was mainly caused by the significant increase of the expression of the curly pilin. The biomembrane-forming ability of the revertant was similar to that of the wild strain. the resistance of the rpoN gene deletion strain to the external environment stress under the condition of acid stress and alkaline stress is remarkably enhanced, This finding is helpful to further study the regulation and control mechanism of the membrane formation of Salmonella typhimurium. in the method of real-time fluorescence quantitative PCR, the difference of the transcription levels of the wild strains of the Salmonella typhimurium and the rpoN gene of the rpoN gene in the biological membrane forming process is determined, the transcription level difference of the cgD, csgA, bcsA, adrA, gcpA, fimA, lpfE and hfq is different, and after the deletion of the rpoN gene, the csgD is detected, The transcription level of csgA gene was significantly enhanced at the time of 4h, 8h and 24h (P0.01), and the expression of gcpA and fimA increased significantly at the 8h level (P0.01), and the expression of lpfE and hfq was significantly enhanced at the 8h transcription level (P0.05), bcsA, There was no significant change in the transcription level of the adrA gene. The adrA, gcpA, fimA and lpfE gene deletion strains were constructed on the basis of the wild strain and the rpoN gene, and the ability of its biological membrane formation was determined. The results showed that the biofilm-forming ability of the single-deletion strain of the adrA gene was significantly lower than that of the wild strain (P0.05), while the loss of gcpA, fimA and lpfE did not have a significant effect on the ability of the membrane to form, and compared with the single deletion of rpoN gene, The biological membrane-forming ability of the double-deleted strain obtained by the deletion of the adrA gene was significantly lower than that of the wild strain (P0.01). The loss of the fimA or fpfE gene results in no significant effect on the ability of the biofilm formation. The expression of rpoS gene and the amount of rpoN gene in rpoS gene were determined by real-time fluorescence quantitative PCR, and the expression of rpoN gene and rpoS gene was not significantly different from the wild strain. That is, there is no significant correlation between the factor RpoN and the RpoS during the film formation of the Salmonella typhimurium. Therefore, the rpoN gene may enhance the formation of the membrane of the Salmonella typhimurium by negative control of the gcpA gene.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
本文编号：2377251
[Abstract]:Salmonella can infect a variety of hosts, resulting in sepsis, gastroenteritis and diarrhea, and can also cause food poisoning in humans, so Salmonella is one of the most important issues affecting global public health and economic development. In the natural environment, Salmonella are present in a suspended state and in a multicell-aggregated colony, such as a biological membrane. The salmonella forms a biological membrane by attachment to the surface of a biological or non-biological surface to resist the identification of the disinfectant, the ambient pressure, the antibiotic and the host immune system, thereby promoting the survival and transmission of bacteria, enhancing its virulence, and related to the outbreak of Salmonella and the continuous infection of the patient. In order to prevent and control the salmonellosis of human and animal, it is necessary to clarify the regulation mechanism of the formation of the biological membrane of Salmonella. By using the Red homologous recombination system to construct the rpoN gene deletion strain and the restoring strain, the strain of Salmonella typhimurium which has stronger ability to form two biofilms is determined by using the Red homologous recombination system, and the expression difference of the biological membrane forming ability and the component is determined, and the RpoN is found to be one of the factors related to the formation of the membrane of the Salmonella typhimurium. in that method, a real-time fluorescence quantitative PCR method is used to measure the difference of the transcription level of the wild strain and the rpoN gene deletion strain of the mouse typhi in the middle and lower reaches of the biological membrane-forming process, On the basis of the deletion of the wild strain and the rpoN gene, a deletion strain was constructed on the basis of the deletion of the wild strain and the rpoN gene, and the ability of its biological membrane formation was determined to further determine the regulation and control network of rpoN gene in the process of membrane formation of Salmonella typhimurium. the construction and biological characteristics of the rpoN gene deletion strain of the Salmonella typhimurium are used to construct the rpoN gene deletion strain by using the Red homologous recombination system to construct the rpoN gene deletion strain by using the Red homologous recombination system, The biological membrane-forming ability and the resistance to environmental stress of the deleted strain and the restoring strain are different, and the expression difference of the biological membrane components is detected by the established csgA and bcsA gene real-time fluorescence quantitative PCR method. The results showed that the loss of rpoN gene was enhanced by the membrane-forming ability as compared with the wild strain, which was mainly caused by the significant increase of the expression of the curly pilin. The biomembrane-forming ability of the revertant was similar to that of the wild strain. the resistance of the rpoN gene deletion strain to the external environment stress under the condition of acid stress and alkaline stress is remarkably enhanced, This finding is helpful to further study the regulation and control mechanism of the membrane formation of Salmonella typhimurium. in the method of real-time fluorescence quantitative PCR, the difference of the transcription levels of the wild strains of the Salmonella typhimurium and the rpoN gene of the rpoN gene in the biological membrane forming process is determined, the transcription level difference of the cgD, csgA, bcsA, adrA, gcpA, fimA, lpfE and hfq is different, and after the deletion of the rpoN gene, the csgD is detected, The transcription level of csgA gene was significantly enhanced at the time of 4h, 8h and 24h (P0.01), and the expression of gcpA and fimA increased significantly at the 8h level (P0.01), and the expression of lpfE and hfq was significantly enhanced at the 8h transcription level (P0.05), bcsA, There was no significant change in the transcription level of the adrA gene. The adrA, gcpA, fimA and lpfE gene deletion strains were constructed on the basis of the wild strain and the rpoN gene, and the ability of its biological membrane formation was determined. The results showed that the biofilm-forming ability of the single-deletion strain of the adrA gene was significantly lower than that of the wild strain (P0.05), while the loss of gcpA, fimA and lpfE did not have a significant effect on the ability of the membrane to form, and compared with the single deletion of rpoN gene, The biological membrane-forming ability of the double-deleted strain obtained by the deletion of the adrA gene was significantly lower than that of the wild strain (P0.01). The loss of the fimA or fpfE gene results in no significant effect on the ability of the biofilm formation. The expression of rpoS gene and the amount of rpoN gene in rpoS gene were determined by real-time fluorescence quantitative PCR, and the expression of rpoN gene and rpoS gene was not significantly different from the wild strain. That is, there is no significant correlation between the factor RpoN and the RpoS during the film formation of the Salmonella typhimurium. Therefore, the rpoN gene may enhance the formation of the membrane of the Salmonella typhimurium by negative control of the gcpA gene.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
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