霍乱弧菌LysR家族基因vc2324及其调控基因vc2323功能的研究

发布时间：2018-02-15 07:45

  本文关键词： 霍乱弧菌 定殖能力 胆盐 竞争黏附 多重抗药性　出处：《南京农业大学》2012年硕士论文　论文类型：学位论文

【摘要】：烈性传染病霍乱由革兰氏阴性细菌霍乱弧菌所引起,该细菌主要毒力基因CTXΦ和VPI(霍乱弧菌毒力岛)受AphB-ToxR级联调控系统调控,该系统中LysR家族基因aphB直接调控霍乱弧菌毒力基因的表达。LysR家族基因广泛存在于细菌各种属中,为原核生物中目前已知最大的转录因子家族,其调控的基因功能多样,涉及合成代谢、群体感应、毒力等众多方面。目前发现霍乱弧菌基因组中约有40个LysR型基因,其中大多尚未被研究。 本文选取其中4个未被研究的LysR型基因分别构建其缺失突变株,通过比较突变株与野生株在定殖能力、毒力、生物膜形成能力等方面的差异,发现了一个参与霍乱弧菌定殖的LysR型基因vc2324。检测其相邻上游基因vc2323的转录情况,证实vc2324正调控vc2323的转录。宿主肠道竞争定殖实验发现vc2323缺失株定殖能力显著下降且较vc2324下降幅度更大。结合LysR家族基因经典调控模式,推测vc2323为影响霍乱弧菌定殖的功能基因。 蛋白质同源性分析发现,假设蛋白VC2323与大肠杆菌中碲抗性蛋白TehA.流感嗜血杆菌HiTehA和拟南芥中SLAC1通道蛋白属于同源蛋白,推测该蛋白可能具有亚碲酸盐抗性、多重耐药性等多种功能。通过TcpA western blot和tcpA转录水平的检测,发现vc2323、vc2324均不影响霍乱弧菌毒力蛋白TCP的表达和形成。检测不同物质对vc2323表达的影响,并比较vc2323缺失株和野生株对KNO2、H2O2的敏感性差异,发现CO2/HCO3-不影响vc2323的表达,vc2323与霍乱弧菌NO/NO2抗性、活性氧抗性无明显关系。同时还发现vc2323、vc2324均不影响霍乱弧菌在LB培养基中生物膜的形成,也与霍乱弧菌对H+耐受力无关。 体外竞争黏附实验发现：粗胆盐存在时,vc2323缺失株较野生株对小鼠小肠的黏附力明显下降,且随黏附时间的延长差异更加显著；而当无粗胆盐存在时,二者对肠道的黏附力无明显差异。且体外实验表明,野生株与vc2323缺失株对粗胆盐的敏感性相同。综合以上结果,分析vc2323对霍乱弧菌定殖能力的影响可能是因为在粗胆盐存在时其能够影响霍乱弧菌对宿主肠道的黏附能力,进而导致vc2323缺失株较野生株定殖能力显著下降,详细的分子机制尚待更深入的研究。 同时,比较不同抗菌物质对缺失株与野生株最小杀菌浓度的差异,发现vc2323缺失株对氯霉素和萘啶酮酸的耐受性分别下降了约64%和74%,对亚碲酸钾的抗性下降了约33%,表明vc2323还参与霍乱弧菌的抗药性和亚碲酸盐抗性。 综上所述,本研究发现LysR家族基因vc2324及其靶基因vc2323参与霍乱弧菌的定殖和抗药性及一定的亚碲酸盐抗性,为霍乱的防控提拱了更多的参考。
[Abstract]:Cholera is caused by Vibrio cholerae, a gram-negative bacterium. The main virulence genes CTX 桅 and virulence island of Vibrio cholerae (Vibrio cholerae) are regulated by AphB-ToxR cascade control system. In this system, LysR family gene aphB directly regulates the expression of virulence gene of Vibrio cholerae. LysR family gene exists widely in various genera of bacteria, and it is the largest known transcription factor family in prokaryotes, and its gene function is diverse. About 40 LysR genes have been found in the genome of Vibrio cholerae, most of which have not been studied. In this paper, four of the unstudied LysR type genes were selected to construct their deletion mutants, and the difference of colonization ability, virulence and biofilm formation ability between the mutant and wild strain was compared. A LysR type gene vc2324was found to be involved in the colonization of Vibrio cholerae. The transcription of its adjacent upstream gene vc2323 was detected. It was confirmed that vc2324 was regulating the transcription of vc2323. The host intestinal competitive colonization assay showed that the colonization ability of vc2323 deficient strains was significantly decreased and the decrease was greater than that of vc2324. Combined with the classical regulation model of LysR family genes, vc2323 was supposed to be the functional gene affecting colonization of Vibrio cholerae. Protein homology analysis showed that the protein VC2323 was assumed to be homologous with the tellurium resistant protein Teha.Haemophilus influenzae HiTehA and Arabidopsis thaliana SLAC1 channel protein, which suggested that the protein might be tellurite resistant. By detecting the transcription level of TcpA western blot and tcpA, it was found that vc2323 and vc2324 did not affect the expression and formation of virulence protein TCP of Vibrio cholerae. The sensitivity of vc2323 deletion strain and wild strain to KNO2H _ 2O _ 2 was compared. It was found that CO _ 2 / HCO _ 3- had no effect on the expression of vc2323 and NO/NO2 resistance of Vibrio cholerae (Vibrio cholerae). It was also found that vc2323 Vc2324 did not affect the biofilm formation of Vibrio cholerae in LB medium and had nothing to do with the tolerance of Vibrio cholerae to H. The results of in vitro competitive adhesion test showed that the adhesion of VC2323 deficient strain to the small intestine of mice was significantly lower than that of the wild strain, and the difference was more significant with the prolongation of the adhesion time, but when there was no crude bile salt in the presence of crude bile salt, the adhesion force of VC2323 strain was significantly lower than that of the wild strain. The results showed that the sensitivity of wild strain to crude bile salt was the same as that of vc2323 deficient strain. The analysis of the effect of vc2323 on colonization ability of Vibrio cholerae may be due to its ability to affect the adhesion of Vibrio cholerae to the intestinal tract of the host in the presence of crude bile salt, which leads to a significant decrease in colonization ability of the vc2323 deficient strain than that of the wild strain. Detailed molecular mechanisms need to be further studied. At the same time, the difference of minimal bactericidal concentration of different antimicrobial substances to the missing strain and the wild strain was compared. It was found that the tolerance to chloramphenicol and naphthalic acid of the vc2323 deletion strain decreased by about 64% and 74, respectively, and the resistance to potassium tellurite decreased by about 33, indicating that vc2323 was also involved in the resistance of Vibrio cholerae and tellurite resistance. In conclusion, this study found that LysR family gene vc2324 and its target gene vc2323 were involved in colonization and drug resistance of Vibrio cholerae and tellurite resistance to some extent, which provided more references for the prevention and control of cholera.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

【共引文献】

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