RpoN對鼠傷寒沙門菌生物被膜形成的調控作用研究
發(fā)布時間：2018-12-13 21:10
【摘要】：沙門菌可感染多種宿主,導致敗血癥、胃腸炎和腹瀉,也可引起人的食物中毒,因此沙門菌病是影響全球公共衛(wèi)生和經(jīng)濟發(fā)展最重要的問題之一。自然環(huán)境中,沙門菌以浮游狀態(tài)和多細胞聚集的菌落如生物被膜狀態(tài)存在。沙門菌通過附著在生物或非生物的表面形成生物被膜,抵抗消毒劑、環(huán)境壓力、抗生素和宿主免疫系統(tǒng)的識別,從而促進細菌生存和傳播,增強其毒力,與沙門菌病的爆發(fā)和患者持續(xù)感染相關。為了更好地防控人和動物的沙門菌病,有必要闡明沙門菌生物被膜形成的調控機制。本研究對兩株生物被膜形成能力較強的鼠傷寒沙門菌利用Red同源重組系統(tǒng)構建rpoN基因缺失株和回復株,測定其生物被膜形成能力及成分的表達差異,發(fā)現(xiàn)RpoN為鼠傷寒沙門菌生物被膜形成相關σ因子之一;運用實時熒光定量PCR的方法測定鼠傷寒沙門菌野生株和rpoN基因缺失株在生物被膜形成過程中下游調控基因的轉錄水平差異,對轉錄水平發(fā)生明顯上調的基因在野生株和rpoN基因缺失株的基礎上分別構建缺失株,測定其生物被膜形成能力,進一步確定rpoN基因在鼠傷寒沙門菌生物被膜形成過程中的調控網(wǎng)絡。1.鼠傷寒沙門菌rpoN基因缺失株的構建及生物學特性研究對兩株生物被膜形成能力較強的鼠傷寒沙門菌利用Red同源重組系統(tǒng)構建rpoN基因缺失株,利用原核表達載體構建回復株;比較野生株、缺失株和回復株的生物被膜形成能力和對環(huán)境應激的抵抗力差異,并通過建立的csgA和bcsA基因實時熒光定量PCR方法檢測了其生物被膜成分的表達差異。結果顯示,與野生株相比,rpoN基因缺失株生物被膜形成能力增強,主要是卷曲菌毛蛋白表達量顯著增加引起的�；貜椭晟锉荒ば纬赡芰εc野生株相似。rpoN基因缺失株在酸性應激和堿性應激條件下對外界環(huán)境應激的抵抗力均顯著增強,說明rpoN基因為鼠傷寒沙門菌中生物被膜形成相關σ因子之一,這一發(fā)現(xiàn)有助于進一步研究沙門菌生物被膜形成的調控機制。2.RpoN在鼠傷寒沙門菌生物被膜形成過程中對下游基因的調控作用以gyrB基因為內參,運用實時熒光定量PCR的方法測定鼠傷寒沙門菌野生株和rpoN基因缺失株在生物被膜形成過程中的下游調控基因csgD、csgA、bcsA、adrA、gcpA、fimA、lpfE、hfq的轉錄水平差異,發(fā)現(xiàn)rpoN基因缺失后,csgD、csgA基因在生物被膜狀態(tài)下4 h、8h和24h這3個時間段轉錄水平均呈現(xiàn)極顯著增強(P0.01),gcpA、fimA基因在8h轉錄水平極顯著提高(P0.01),lpfE、hfq基因也在8h轉錄水平有顯著增強(P0.05),bcsA、adrA基因轉錄水平卻沒有顯著變化。在野生株和rpoN基因缺失株的基礎上分別構建adrA、gcpA、fimA、lpfE基因缺失株,測定其生物被膜形成能力。結果顯示,與野生株相比,adrA基因單缺失株的生物被膜形成能力顯著下降(P0.05),而gcpA、fimA、lpfE基因缺失后對其生物被膜形成能力沒有明顯的影響;與rpoN基因單缺失相比,在其基礎上缺失adrA基因得到的雙缺失株生物被膜形成能力顯著下降(P0.05),缺失gcpA基因得到的雙缺失株生物被膜形成能力極顯著下降(P0.01),與野生株相似。缺失fimA或fpfE基因得到的雙缺失株生物被膜形成能力沒有明顯的影響。通過實時熒光定量PCR測定兩株鼠傷寒沙門菌rpoN基因單缺失株中rpoS基因的表達量以及rpoS基因單缺失株中rpoN基因的表達量,與野生株相比,rpoN基因和rpoS基因的表達量均沒有顯著差異,即在鼠傷寒沙門菌生物被膜形成過程中σ因子RpoN與RpoS之間沒有明顯的相關性。因此,rpoN基因可能通過負調控gcpA基因增強鼠傷寒沙門菌生物被膜的形成。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R378
本文編號：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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R378
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