铜绿假单胞菌PA3114基因的调控功能及亚抑菌浓度壮观霉素对phzA2激活效应的研究

发布时间：2018-04-22 02:01

本文选题：铜绿假单胞菌 + 假尿嘧啶合成酶　；参考：《西北大学》2016年硕士论文

【摘要】：铜绿假单胞菌是医院内感染的三大致病菌之一,可以引起各种感染,从轻微的皮肤感染到严重的导致病人死亡。和其他致病菌一样,铜绿假单胞菌的致病能力主要取决于其拥有的作用于宿主的多种致病因子。而这些致病因子基因受复杂的调控系统调节,使铜绿假单胞菌适应宿主环境条件的变化。吩嗪化合物绿脓菌素作为铜绿假单胞菌分泌的次级代谢产物,可以作为抗生素抑制其他细菌的生长。同时,绿脓菌素也是铜绿假单胞菌重要的致病因子,还可以作为信号分子调节众多基因的表达。在铜绿假单胞菌中,吩嗪化合物的合成主要由phzl(phzA1B1C1D1E1F1G1)以及phz2 (phzA2B2C2D2E2F2G2)两个基因簇来完成。实验室前期发现,25μg/mL浓度的壮观霉素在不抑菌的情况下会激活基因簇phzl,phz2的表达,在筛选与激活作用相关的基因时发现PA3114突变后,铜绿假单胞菌基因phzA2的表达明显降低,且壮观霉素激活效应也显著减弱。本课题针对PA3114基因的调控功能进行了研究,探究了PA3114与phzA2基因表达之间的关系,以及PA3114调控的可能机理和途径。除此之外,还初步探究了亚抑菌浓度壮观霉素对铜绿假单胞菌基因的激活作用机理。研究首先构建了PA3114敲除突变体PAO1 (△PA3114)及互补体PAO1 (APA3114 pAK3114)。结果表明PA3114突变后,相对于野生菌PAO1,菌株中吩嗪合成相关基因phzA1, phzA2以及phzS的表达明显降低,且绿脓菌素的产量明显减少。由于吩嗪物质的合成受PQS(Pseudomonas quinolone signal)调节,实验对PA3114与PQS之间的关系进行了研究,结果显示突变体中pqsA,pqsR以及pqsH的基因表达明显降低,且PQS的产量也明显减少。这表明PA3114可能通过影响PQS而进一步影响绿脓菌素的生成。进一步研究发现,突变体PAO1 (△PA3114)中三型分泌系统的效应基因exoT, exoS,exoY的基因表达也明显降低。同时,PAO1(△PA3114)菌株的蹭行运动能力几乎消失,丛动能力明显增强,生物膜的产量明显减少；而电镜结果显示PAO1(△PA3114)菌株的鞭毛没有变化但是菌毛数目大量减少。研究发现,突变体中rsmA表达降低,rsmY, rsmZ表达明显升高,说明PA3114突变后,可能通过Rsm途径对铜绿假单胞菌体内多个基因进行调控,导致突变体中三型分泌蛋白、运动能力以及绿脓菌素的合成发生变化。对亚抑菌浓度的壮观霉素激活作用的研究发现,PA3114突变后,菌体的壮观霉素亚抑菌浓度与野生型PAO 1一致。进一步研究发现,壮观霉素浓度越大对基因phzA2的激活作用越强。除此之外,亚抑菌浓度的壮观霉素对铜绿假单胞菌phzS, phzA1, pilG等基因都有激活作用。PA3114突变后,亚抑菌浓度的壮观霉素对铜绿假单胞菌基因的激活效应依然存在,但是程度明显减弱。壮观霉素对基因phzA2激活的具体机制有待进一步的研究。
[Abstract]:Pseudomonas aeruginosa is one of the three major nosocomial infections that can cause infections ranging from mild skin infections to severe death. The pathogenicity of Pseudomonas aeruginosa, like other pathogenic bacteria, is mainly determined by its multiple host factors. These genes are regulated by complex regulatory systems, which make Pseudomonas aeruginosa adapt to the changes of host environment. As a secondary metabolite of Pseudomonas aeruginosa, phenazine compounds can inhibit the growth of other bacteria as antibiotics. Meanwhile, Pseudomonas aeruginosa is an important pathogenic factor of Pseudomonas aeruginosa and can regulate the expression of many genes as signal molecules. In Pseudomonas aeruginosa, the synthesis of phenazine compounds was mainly carried out by two gene clusters, phzlzA1B1C1D1E1F1G1) and phz2 phzA2B2B2D2E2F2G2). It was found that spectinomycin at 25 渭 g/mL concentration in laboratory could activate the expression of phzlz2 gene cluster in the absence of bacteriostasis. The phzA2 expression of Pseudomonas aeruginosa gene was significantly decreased after PA3114 mutation was found in screening genes related to activation. The activation effect of spectinomycin was also significantly weakened. In this paper, the regulatory function of PA3114 gene was studied, the relationship between PA3114 and phzA2 gene expression, and the possible mechanism and pathway of PA3114 regulation were explored. In addition, the activation mechanism of spectinomycin on Pseudomonas aeruginosa gene was studied. Firstly, the PA3114 knockout mutant PAO1 (PA3114) and the complementary mutant PAO1 A3114pAK3114 were constructed. The results showed that the expression of phenazine synthesis-related genes phZA1, phzA2 and phzS were significantly decreased after PA3114 mutation, and the yield of Pseudomonas aeruginosa was significantly decreased compared with the wild strain PAO1. Since the synthesis of phenazine was regulated by PQS(Pseudomonas quinolone signal, the relationship between PA3114 and PQS was studied. The results showed that the gene expression of pqsAgna pqsR and pqsH in the mutant was significantly decreased, and the yield of PQS was also significantly decreased. This suggests that PA3114 may further affect the production of Pseudomonas aeruginosa by affecting PQS. Further studies showed that the gene expression of exoT and exoSnexoY in the secretory system of the mutant PAO1 (PA3114) was also significantly decreased. At the same time, the motility of PAO1 (PA3114) strain almost disappeared, the ability of cluster motility increased obviously, and the yield of biofilm decreased obviously, while the electron microscopic results showed that the flagella of PAO1 (PA3114) strain had no change, but the number of fimbriae decreased significantly. It was found that the expression of rsmA decreased and the expression of rsmY and rsmZ increased significantly in the mutant, which indicated that after PA3114 mutation, multiple genes in Pseudomonas aeruginosa might be regulated by Rsm pathway, leading to the secretion of three types of protein in the mutant. Motor ability and the synthesis of Pseudomonas aeruginosa changed. Studies on the activation of spectinomycin in subinhibitory concentration showed that the subinhibitory concentration of spectinomycin was consistent with that of wild type PAO 1 after mutation of PA3114. Further studies showed that the higher the concentration of spectinomycin, the stronger the activation of gene phzA2. In addition, the subinhibitory concentration of spectinomycin could activate the genes of Pseudomonas aeruginosa, such as phzS, phzA1, pilG and so on. After the mutation of PA3114, the effect of spectinomycin of subinhibitory concentration on the gene activation of Pseudomonas aeruginosa still existed, but the degree was obviously weakened. The specific mechanism of spectinomycin on gene phzA2 activation needs further study.
【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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