铜绿假单胞菌中两个调节子基因对致病因子的调控研究

发布时间：2018-01-24 02:31

本文关键词： 铜绿假单胞菌调节子 Ⅲ型分泌系统致病因子蛋白定位　出处：《西北大学》2016年硕士论文　论文类型：学位论文

【摘要】：铜绿假单胞菌(Pseudomonas aerugionsa, PA)是广泛存在于自然界中的革兰氏阴性杆菌,它可以感染人类引起不同病症甚至造成死亡。铜绿假单胞菌之所以普遍存在、适应性强受益于其体内有多种基因调控系统,能快速调节基因表达以应对外界条件变化。Gac/Rsm系统是铜绿假单胞菌中一个重要的基因调控系统,其小蛋白RsmA是一种全局调控因子,调控多种致病因子的表达。本实验室在前期筛选rsmA调控基因时,得到一个影响rsmA表达的基因PA4664 (prmC)。大肠杆菌中与PA4664同源的基因prmC是一个肽链释放因子的甲基转移酶,参与肽链合成的正常释放。本课题以突变体/△PA4664为研究对象,研究了PA4664基因对铜绿假单胞菌多种表型的影响及其对相关基因调控的分子机制。研究发现,突变体△PA4664的泳动、丛动和蹭行运动均明显降低。在突变体△PA4664中互补rsmA可使运动的表型恢复至野生型水平。然而,电镜观察突变体△PA4664中的鞭毛和菌毛并未明显受损,说明PA4664对运动的影响并不是通过影响鞭毛和菌毛的结构,而是通过调节RsmA实现的。实验发现,除运动能力外,PA4664还影响铜绿假单胞菌群体感应系统(Quorum sensing, QS)以及生物被膜的生成。在突变体△PA4664中,QS系统的las和rhl基因表达量都明显降低,生物被膜多聚复合物合成量减少；突变体△PA4664胞内c-di-GMP也降低了近一半。同时PA4664也影响菌体的致病因子,如绿脓菌素和T3SS分泌蛋白。以果蝇(Drosophila melanogaster Canton S)和中国白菜(Brassica pekinensis)为模型的致病性实验表明,突变体/△PA4664的致病性相对于野生型有较大降低。在本研究构建的突变体/△PA4664的互补体中,运动性、绿脓菌素产量和c-di-GMP水平等均恢复至野生型水平,进一步证实PA4664调控作用。综上所述,这些结果表明,PA4664是rsmA上游调控网络的一分子,它通过rsmA对铜绿假单胞菌的运动性等表型起重要的调控作用。本研究同时对PA4595蛋白进行初步研究。亚细胞定位免疫印迹实验证明PA4595蛋白属于ATP结合盒ABC蛋白家族的REG亚家族。REG亚家族蛋白定位于细胞质中,与普通的ABC蛋白定位在生物膜上不同,不参与物质运输。PA4595基因突变后T3SS的分泌蛋白和调节蛋白表达均升高,本研究发现它并不是通过vfr或者rsmA等对ExsA的调节而导致以上变化的。
[Abstract]:Pseudomonas aerugionsa (PA) is a gram-negative bacilli widely found in nature. Pseudomonas aeruginosa is widely found in humans because of its adaptability to a variety of gene regulatory systems. Gac / Rsm system is an important gene regulation system in Pseudomonas aeruginosa, and its small protein RsmA is a global regulatory factor. Regulation of the expression of multiple pathogenic factors. Our laboratory screening of rsmA regulatory genes in the early stage. A gene that affects the expression of rsmA was obtained. The gene prmC, which is homologous to PA4664 in E. coli, is a methyltransferase of peptide chain releasing factor. Participate in the normal release of peptide chain synthesis. In this study, the mutant / PA4664 as the research object. The effects of PA4664 gene on many phenotypes of Pseudomonas aeruginosa and the molecular mechanism of regulation of related genes were studied. In the mutant PA4664, complementary rsmA could restore the exercise phenotype to the wild-type level. Electron microscopic observation showed that the flagella and fimbriae in the mutant PA4664 were not significantly damaged, indicating that the effect of PA4664 on the movement was not by affecting the structure of the flagella and the fimbriae. The results showed that PA4664 also affected the colony sensing system of Pseudomonas aeruginosa (Pseudomonas aeruginosa), Quorum sensing. QS) and biofilm formation. The expression of las and rhl genes in the QS system of mutant PA4664 was significantly decreased, and the synthesis of biofilm polycomplex was decreased. The intracellular c-di-GMP of the mutant PA4664 was reduced by nearly half, and PA4664 also affected the pathogenic factors of the bacteria. For example, Pseudomonas aeruginosa and T3SS secreted proteins. Drosophila melanogaster Canton S) and Chinese cabbage (. Brassica pekinensis is the pathogenicity of the model. The pathogenicity of mutants / PA4664 was significantly lower than that of wild type. In the mutants / PA4664 complementants constructed in this study, motility was observed. Pseudomonas aeruginosa yield and c-di-GMP level returned to the wild-type level, further confirmed the regulatory role of PA4664. In conclusion, these results show that. PA4664 is a member of rsmA upstream regulatory network. It plays an important role in regulating the motility and other phenotypes of Pseudomonas aeruginosa by rsmA. In this study, we also studied the PA4595 protein. Subcellular localization immunoblotting assay showed that PA4595. The protein belongs to the REG subfamily of the ATP binding box ABC family. The protein of the REG subfamily is located in the cytoplasm. The expression of T3SS secretory protein and regulatory protein were increased after the mutation of PA4595 gene, which was different from the normal ABC protein in biofilm. This study found that these changes were not caused by the regulation of ExsA by vfr or rsmA.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R378

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