霍乱弧菌群体感应调控因子VqmA活性相关基因的筛选及LysR和MFS蛋白功能的研究

发布时间：2018-01-05 18:22

本文关键词：霍乱弧菌群体感应调控因子VqmA活性相关基因的筛选及LysR和MFS蛋白功能的研究　出处：《南京农业大学》2010年硕士论文　论文类型：学位论文

【摘要】：霍乱弧菌(V. cholerae)是一种革兰氏阴性、兼性厌氧小杆菌,是引起人类腹泻疾病霍乱的病原体。大量研究证明,霍乱弧菌的致病能力主要是由霍乱毒素(cholerae toxin, CT)和毒素共调节菌毛(toxin-coregulated pilus, TCP)引起的。霍乱弧菌毒力基因的表达受到各种环境条件的影响,如营养物浓度和种类、温度、pH、胆汁盐和群体感应信号分子。 HapR是霍乱弧菌群体感应调控系统的关键调控因子,可以抑制霍乱弧菌CT和TCP表达。LuxR家族蛋白VqmA可以在低细胞浓度时诱导HapR的表达从而降低细菌的致病性。本文通过构建转座子随机突变文库的方法筛选影响VqmA活性的基因,获得七株候选的突变株,并且对突变株M1(△VC0073, SAM-依赖的甲基转移酶)做了深入的研究。研究发现该基因缺失后,VqmA无法激活报告基因的表达,并且VqmA的转录调控活性随着该基因的回复表达而恢复,VC0073是VqmA发挥活性的必须基因。VC0073编码合成的蛋白与SAM-依赖的甲基转移酶的同源性最高,在很多细菌中该酶参与一些小分子化合物的合成,VqmA激活目的基因的过程可能需要该酶合成的某类化合物的协助,但是研究发现该化合物并不存在于细菌上清液中。 LysR类蛋白是一种全局性的转录调控因子,霍乱弧菌中约有40个LysR类型的蛋白。借助生物信息学工具,在霍乱弧菌中发现了五类比较特殊的LysR蛋白,这些蛋白的下游都为MFS家族蛋白。MFS家族蛋白属于多重药物转运蛋白。我们选取其中一组基因VC1617 (Encoding LysR Protein)和VC1618 (Encoding Multidrug Resistance protein)作为研究对象,研究发现VC1617缺失和过表达后霍乱弧菌可以在LB和M9培养基正常生长,VC1617并不是细菌在这两中培养基生长的必需基因,这在一定程度上也反映出了LysR蛋白生理功能的多样性。通过构建Luminescence检测质粒发现LysR不但可以抑制自身的表达而且还可以激活下游基因VC1618 (Multidrug Resistance Gene)的表达。Western blot试验表明VC1617缺失后霍乱弧菌毒力基因的表达并没有受到影响；虽然VC1 617缺失后多重药物转运蛋白VC1618的表达量大为降低,但是突变株对一些特定药物的耐受能力并没有削弱；在毒力基因的表达量和药物的耐受能力都没有降低的情况下,突变株在小鼠体内的定殖能力也没有降低。
[Abstract]:Vibrio cholerae (V. cholerae) is a gram negative, facultative anaerobic bacteria is small, causing cholera diarrhea disease human pathogens. Many studies have demonstrated that Vibrio cholerae pathogenicity is mainly caused by cholera toxin (cholerae toxin, CT) and the toxin coregulated pilus (toxin-coregulated pilus, TCP). The influence of expression caused by Vibrio Cholera toxin genes are subjected to various environmental conditions, such as the type and concentration of nutrients, temperature, pH, bile salt and quorum sensing signal.
HapR is a key regulator of V.cholerae quorum sensing regulation system, can inhibit Vibrio cholerae CT and TCP expression of.LuxR family protein VqmA can induce HapR cells at low concentration so as to reduce pathogenic bacteria. The construction method of transposon random mutation library screening influence the activity of VqmA gene, seven strains of candidate the mutant strain, and the mutant M1 (VC0073, SAM- dependent methyl transferase) were studied. The study found that the gene deletion, VqmA could not activate the reporter gene expression, transcriptional regulation and VqmA control activity restored along with the expression of this gene is VqmA VC0073 reply, the activity must play methyl.VC0073 protein and SAM- gene encoding synthesis dependent transferase homology in many bacteria, the enzymes involved in some small molecular compounds synthesis, activation of VqmA gene The process may require the assistance of some of the compounds synthesized by the enzyme, but the study found that the compound does not exist in the bacterial supernatant.
LysR protein is a global transcriptional regulator, Vibrio cholerae has about 40 types of LysR protein. Using bioinformatics tools, five kinds of special LysR protein was found in Vibrio cholerae, these proteins are downstream of the MFS protein family.MFS family proteins belong to multiple drug transporters. We selected a group of genes VC1617 (Encoding LysR Protein) and VC1618 (Encoding Multidrug Resistance protein) as the research object, the study found that deletion of VC1617 and overexpression of Vibrio cholerae can grow normally in the culture medium of LB and M9, VC1617 is not the genes required for the growth of bacterial culture in two, which to some extent also reflects the diversity of LysR protein physiological function. By constructing the Luminescence plasmid detection found LysR not only inhibits its own expression but also can activate downstream gene VC1618 (Mu Ltidrug Resistance Gene.Western blot) expression test showed that the expression of virulence genes of Vibrio cholerae VC1617 deletion was not affected; while the expression of VC1 617 after deletion of multiple drug transporter VC1618 is greatly reduced, but the mutant for certain drug resistance ability has not weakened; in the expression of virulence genes and the amount of drug tolerance has been reduced under the condition of the mutant colonization on the mice are not reduced.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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