霍乱弧菌调控aphB的基因筛选及功能研究及铜绿假单胞菌对霍乱弧菌抑制作用研究
发布时间：2018-07-28 19:08
【摘要】：霍乱是一种急性腹泻疾病,由革兰氏阴性细菌霍乱弧菌(Vibrio cholerae)引起。通常是血清群01群的古典型和埃尔托生物型霍乱弧菌致病,临床表现为大量米泔样大便,严重时候导致病人产生霍乱肌无力并且迅速脱水。如果迅速和适当的治疗没有跟进,可导致病人死亡。 毒素协同调节菌毛(the toxin-coregulated pilus, TCP)是霍乱感染中的重要毒力因子,能协助霍乱弧菌定殖于小肠。霍乱弧菌产生的霍乱毒素(cholera toxin, CT)则导致病人剧烈腹泻。负责合成TCP的基因以操纵子的方式位于霍乱弧菌基因组中一个大毒力岛上,被称做TCP-ACF元件或霍乱毒力岛(vibrio pathogenicit island,VPI).CT毒素是由ctxA和ctxB两个基因编码,它们位于另一个遗传元件原性噬菌体CTXΦ上。而位于霍乱毒力岛上的ToxT是AraC型调控子,能直接激活tcp,ctxAB和其他辅助定殖因子的表达。ToxT的表达则是依赖于两组基因的合作,包括大染色体上的跨膜调控蛋白编码基因toxRS和霍乱毒力岛上的tcpPH。而AphB与AphA协同作用可以激活tcpPH的表达,同时也是导致两大霍乱致病型古典型和埃尔托生物型毒力基因表达差异的原因。 霍乱弧菌有三套平行的群体感应系统,共同调控霍乱毒力基因表达。研究证明,霍乱弧菌群体感应系统通过关键调控蛋白HapR负调控毒力基因的表达.HapR通过直接作用于aphA的启动子区调控毒力级联系统,降低毒力的表达。但是HapR并不影响aphB的表达。为了研究aphB的表达调控机理,我们利用转座子插入突变建库的方法进行筛选。我们将aphB启动子区克隆到两个报告质粒pBBRLux和pKP302上,并将其导入埃尔托生物型霍乱弧菌C6706(lacZ-)中,以此作为出发菌株。利用出发菌株与转座子pSC123接合建库,通过检测aphB启动子的表达水平来筛选影响aphB表达的突变株。在本研究中,我们筛选到两株突变株T1和T2能影响aphB的表达。运用随机扩增PCR方法检测转座子插入位点,通过测序比对分析后发现T1中转座子插入在vc1585读码框内,而T2中转座子则插入在距vc1602基因末端7bp处。通过分别构建框内缺失株,我们确定vc1585能够影响aphB的表达,而vc1602及vc1601单缺失并不影响aphB的表达。 在另一个实验中我们拟筛选能够抑制霍乱弧菌C6706生长的细菌。经过筛选我们发现了十株细菌能显著抑制霍乱弧菌的生长。经API20E试纸条鉴定,发现十株菌均为铜绿假单胞菌(Pseudomonas aeruginosa)。通过与铜绿假单胞菌标准菌株ATCC27853对比发现可能是绿脓素(Pyocyanin, PCN)在抑菌过程中起主要作用。
[Abstract]:Cholera is an acute diarrhea disease caused by Vibrio cholerae (Vibrio cholerae). Vibrio cholerae, usually an ancient and biologic serotype of serogroup 01, is clinically characterized by a large amount of hogwash stool, which in severe cases leads to cholera myasthenia and rapid dehydration. If prompt and appropriate treatment is not followed up, the patient may die. Toxin coregulatory pili (the toxin-coregulated pilus, TCP) is an important virulence factor in cholera infection, which can help Vibrio cholerae colonize the small intestine. The cholera toxin (cholera toxin, CT) produced by Vibrio cholerae causes severe diarrhea. The genes responsible for the synthesis of TCP are located in a virulent island in the Vibrio cholerae genome in the form of manipulators, known as TCP-ACF elements or (vibrio pathogenicit islandVPI) .CT toxins are encoded by two genes, ctxA and ctxB. They are located on another genetic element, CTX 桅. ToxT, located on cholera virulence island, is a AraC type regulator, which can directly activate the expression of tcpnctxAB and other auxiliary colonizing factors. The expression of ToxT depends on the cooperation of two groups of genes. The transmembrane regulatory protein encoding gene toxRS and the cholera virulence island tcpPH. The synergistic action of AphB and AphA can activate the expression of tcpPH, and it is also the reason for the difference of virulence gene expression between the two major pathogenic types of cholera, paleotypic and Elto. Vibrio cholerae has three sets of parallel population sensing systems to regulate cholera virulence gene expression. The results showed that the virulence cascade system of Vibrio cholerae could reduce the expression of virulence genes by directly acting on the promoter region of aphA through the negative regulation of virulence gene expression of the key regulatory protein HapR by the population sensing system of Vibrio cholerae (Vibrio cholerae). But HapR does not affect the expression of aphB. In order to study the expression regulation mechanism of aphB, we used transposon insertion mutation to construct library. The aphB promoter region was cloned into two reporter plasmids pBBRLux and pKP302 and introduced into Vibrio cholerae C6706 (lacZ-). The library was constructed by the conjugation of the original strain and transposon pSC123, and the expression level of aphB promoter was detected to screen the mutants that affected the expression of aphB. In this study, we selected two mutant strains T 1 and T 2 to influence the expression of aphB. The insertion site of transposon was detected by random amplified PCR. The results showed that T1 transposon was inserted into vc1585 reading frame, while T2 transposon was inserted at the end of vc1602 gene 7bp. We determined that vc1585 could affect the expression of aphB, while vc1602 and vc1601 single deletion did not affect the expression of aphB. In another experiment, we intended to screen bacteria that could inhibit the growth of Vibrio cholerae C 6706. After screening, we found that ten strains of bacteria could significantly inhibit the growth of Vibrio cholerae. Ten strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa). Were identified by API20E test strip. Compared with the standard Pseudomonas aeruginosa strain ATCC27853, it was found that the pyocyanin (Pyocyanin, PCN) may play a major role in the inhibition of Pseudomonas aeruginosa.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
[Abstract]:Cholera is an acute diarrhea disease caused by Vibrio cholerae (Vibrio cholerae). Vibrio cholerae, usually an ancient and biologic serotype of serogroup 01, is clinically characterized by a large amount of hogwash stool, which in severe cases leads to cholera myasthenia and rapid dehydration. If prompt and appropriate treatment is not followed up, the patient may die. Toxin coregulatory pili (the toxin-coregulated pilus, TCP) is an important virulence factor in cholera infection, which can help Vibrio cholerae colonize the small intestine. The cholera toxin (cholera toxin, CT) produced by Vibrio cholerae causes severe diarrhea. The genes responsible for the synthesis of TCP are located in a virulent island in the Vibrio cholerae genome in the form of manipulators, known as TCP-ACF elements or (vibrio pathogenicit islandVPI) .CT toxins are encoded by two genes, ctxA and ctxB. They are located on another genetic element, CTX 桅. ToxT, located on cholera virulence island, is a AraC type regulator, which can directly activate the expression of tcpnctxAB and other auxiliary colonizing factors. The expression of ToxT depends on the cooperation of two groups of genes. The transmembrane regulatory protein encoding gene toxRS and the cholera virulence island tcpPH. The synergistic action of AphB and AphA can activate the expression of tcpPH, and it is also the reason for the difference of virulence gene expression between the two major pathogenic types of cholera, paleotypic and Elto. Vibrio cholerae has three sets of parallel population sensing systems to regulate cholera virulence gene expression. The results showed that the virulence cascade system of Vibrio cholerae could reduce the expression of virulence genes by directly acting on the promoter region of aphA through the negative regulation of virulence gene expression of the key regulatory protein HapR by the population sensing system of Vibrio cholerae (Vibrio cholerae). But HapR does not affect the expression of aphB. In order to study the expression regulation mechanism of aphB, we used transposon insertion mutation to construct library. The aphB promoter region was cloned into two reporter plasmids pBBRLux and pKP302 and introduced into Vibrio cholerae C6706 (lacZ-). The library was constructed by the conjugation of the original strain and transposon pSC123, and the expression level of aphB promoter was detected to screen the mutants that affected the expression of aphB. In this study, we selected two mutant strains T 1 and T 2 to influence the expression of aphB. The insertion site of transposon was detected by random amplified PCR. The results showed that T1 transposon was inserted into vc1585 reading frame, while T2 transposon was inserted at the end of vc1602 gene 7bp. We determined that vc1585 could affect the expression of aphB, while vc1602 and vc1601 single deletion did not affect the expression of aphB. In another experiment, we intended to screen bacteria that could inhibit the growth of Vibrio cholerae C 6706. After screening, we found that ten strains of bacteria could significantly inhibit the growth of Vibrio cholerae. Ten strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa). Were identified by API20E test strip. Compared with the standard Pseudomonas aeruginosa strain ATCC27853, it was found that the pyocyanin (Pyocyanin, PCN) may play a major role in the inhibition of Pseudomonas aeruginosa.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378
【共引文献】
相关期刊论文 前1条
1 田辉;;肠道致病菌群体感应研究进展[J];世界华人消化杂志;2007年08期
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1 史晓，
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