铜绿假单胞菌PAO1的酰基转移酶编码基因PA3242的功能研究

发布时间：2018-01-29 22:47

  本文关键词： 铜绿假单胞菌 类脂A 生长 抗生素抗性 毒性　出处：《西北大学》2017年硕士论文　论文类型：学位论文

【摘要】：铜绿假单胞菌是一种常见的临床机会致病菌,引起了全球约10%的临床感染。感染该类病菌的患者通常免疫力低下,且致死率极高。另外在治疗过程中,该类致病菌会迅速对抗生素类药物产生抗性,针对该种病原菌感染的治疗形势不容乐观。铜绿假单胞菌拥有多种抗性机制,其中外膜的低渗透性是其产生高耐药性的主要因素之一。外膜外侧的主要结构为脂多糖(LPS),该物质由类脂A、核心寡糖和O抗原构成,其中类脂A不仅是内毒素活性成分,同时还将脂多糖(LPS)紧密地锚定在外膜上,有效地维持了外膜的稳定性与完整性。近年有关于类脂A的研究表明:该分子可影响多种病原菌的生长、抗生素抗性、毒性以及免疫原性等诸多生理特性。实验室前期发现PA0011基因的缺失可显著影响PAO1的抗生素抗性与毒性,据PAO1官方网站推测,该基因为类脂A酰基转移酶HtrB同源基因。将HtrB与PAO1基因组进行比对发现另一个基因PA3242。该基因与HtrB的具有高达47%的同源性,预测其与PA0011共同参与PAO1中类脂A结构的二级酰基化修饰。本课题以PA3242为主要研究对象,深入探究了该基因对PAO1生长、抗生素抗性和毒性等诸多生理特性的影响,同时与PA0011进行了比较,对PAO1中酰基转移酶的作用机理有了更为深刻的认识。PA3242对PAO1的生长影响极大,该基因的缺失导致突变体PAO1(△PA3242)在37℃的生长严重受限,且无法在温度低于28℃的条件下存活,但PA0011的缺失却对PAO1的生长没有任何影响。PA3242的转录水平随温度降低而升高,其在28℃下的转录水平是37℃条件下的5倍。虽然PA0011在低温下的表达也有所升高,但表达水平远远低于PA3242。以上结果表明:PA3242对于菌体在低温下的生存起到更为重要的作用。另外,PAO1中类脂A翻转酶基因PA4997的过表达可恢复突变体PAO1(△PA3242)的生长,表明PA3242很可能参与到类脂A的转运。PA3242的缺失会严重破坏外膜的完整性与稳定性并进一步影响PAO1的抗生素抗性。由实验可知:虽然PA0011与PA3242的缺失均导致PAO1抗生素敏感性降低,但与PA0011相比,PA3242的缺失导致PAO1对更多种类的抗生素表现出更高的敏感性,同时对金属螯合剂EDTA的耐受力也大大降低。PA3242还可显著影响PAO1的毒性。与PAO1和PAO1(△PA0011)相比,PAO1(△PA3242)的细胞毒性和致病性都显著减弱。实验表明:PA3242的缺失导致PAO1外膜畸变并丢失所有菌毛结构,而突变体PAO1(△PA0011)仍保有部分菌毛并具有规则的外膜结构。进一步研究发现,突变体PAO1(△PA3242)丧失了丛动、泳动和蹭行能力,而突变体PAO1(△PA0011)仅从动能力部分受限。由以上结论可知:与PA0011相比,PA3242不仅能够显著影响PAO1抗生素抗性和毒性,它还极有可能参与到类脂A转运过程,并进一步影响PAO1的生长、菌体形态和菌毛装配。表明该基因在二级酰基化修饰过程中起主导作用。以该基因为治疗靶点可为治疗铜绿假单胞菌感染提供许多新的思路。
[Abstract]:Pseudomonas aeruginosa is a common clinical opportunistic pathogen that causes about 10% clinical infections worldwide. Patients infected with Pseudomonas aeruginosa usually have low immunity and high mortality. This kind of pathogenic bacteria can quickly develop resistance to antibiotics, and the treatment of the pathogen infection is not optimistic. Pseudomonas aeruginosa has many resistance mechanisms. The low permeability of outer membrane is one of the main factors to produce high drug resistance. The main structure of outer membrane is lipopolysaccharide (LPS), which is composed of lipopolysaccharide, core oligosaccharide and O antigen. Lipids A is not only the active component of endotoxin, but also the lipopolysaccharide (LPS) is closely anchored on the outer membrane. It can effectively maintain the stability and integrity of the outer membrane. Recent studies on lipids A have shown that this molecule can affect the growth of many pathogens and antibiotic resistance. Toxicity, immunogenicity and many other physiological characteristics. The lack of PA0011 gene can significantly affect the antibiotic resistance and toxicity of PAO1, according to the PAO1 official website. This gene is based on the HtrB homologous gene of lipoacyltransferase. Another gene PA3242has been found by comparing the genome of HtrB and PAO1. The gene has a 47% homology with HtrB. Source. It is predicted that PA3242 and PA0011 are involved in the secondary acylation modification of liposome A structure in PAO1. In this study, PA3242 was used as the main research object to explore the growth of PAO1 by the gene. The effects of antibiotic resistance and toxicity on many physiological characteristics were also compared with those of PA0011. The effect of PA3242 on the growth of PAO1 was greatly influenced by a deeper understanding of the mechanism of acyltransferase in PAO1. The deletion of the gene caused the mutant PAO1 (PA3242) to grow at 37 鈩，

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