沙门菌密度感应系统对六型分泌系统调控的相关研究
发布时间：2018-10-13 18:59
【摘要】：沙门菌是一种重要的人畜共患病原菌,在自然界分布广泛。绝大多数沙门菌对人和动物都有致病性,能引起人和动物多种不同临床表现的沙门菌病。沙门菌分为肠道沙门菌和邦戈沙门菌两个种。沙门菌血清型众多,大约拥有2500多种血清型,其中肠炎沙门菌和鼠伤寒沙门菌在人类食源性沙门菌病中尤为重要。六型分泌系统(Type 6 secretion system,T6SS)是近年来发现的革兰阴性菌中一种新的蛋白转运系统,它可将蛋白质运送到胞外。T6SS广泛存在于细菌中,其结构组成包括Icm F家族、ATP酶-Clp V、Fha调控蛋白以及分泌相关的Vgr G和Hcp蛋白。目前发现T6SS在细菌对外界环境的适应性、对宿主细胞的致病力、生物被膜形成等方面发挥作用。与其他分泌系统一样,T6SS的转录和表达水平也受到严格调控的。有研究证实,在霍乱弧菌中密度感应(Quorum sensing,QS)系统lux S基因能显著影响T6SS主要元件的转录水平。因此,本课题通过构建沙门菌lux S基因缺失株,研究沙门菌中QS对T6SS的影响。1.沙门菌毒力因子多重PCR检测方法的建立及应用通过建立沙门菌不同毒力岛基因的多重PCR方法,对沙门菌毒力基因进行快速、灵敏的检测。根据Gen Bank序列设计合成16对引物,优化反应条件,建立四组多重PCR方法。利用建立的多重PCR方法对124株沙门菌进行毒力基因检测,结果显示除了sop E基因分布率为15%外,其它15个毒力基因分布率可达90%以上。选取代表性菌株,测定其对小鼠的致病力,结果显示沙门菌均可导致小鼠发病死亡。药敏试验结果显示沙门菌对红霉素、阿奇霉素、克林霉素、新霉素、大观霉素、四环素、利福平等抗生素的耐药率可达90%以上,且存在多重耐药性。2.沙门菌lux S基因缺失株的构建及生物学特性研究利用Red同源重组系统构建沙门菌lux S基因缺失株,并对其生长速度、运动性、生物被膜形成、致病力以及对鼠巨噬细胞RAW264.7黏附入侵等生物学特性进行研究。结果显示,Lux S对沙门菌的生长速度、生物被膜形成能力、致病力及细胞黏附侵袭能力均无明显影响。但lux S缺失株的运动性显著高于野生株。3.密度感应系统Lux S蛋白对T6SS影响的分析克隆表达并纯化T6SS核心组分Hcp1、Hcp2和Clp V蛋白,制备相应的免疫血清。应用荧光定量PCR以及免疫印迹技术对lux S基因缺失株中hcp1、hcp2和clp V的基因转录水平和蛋白表达水平进行检测。结果显示,无论在对数生长期和平台期,lux S基因缺失株与野生株中T6SS核心组分的转录水平和表达水平均无明显差异。本研究表明QS系统Lux S蛋白并不影响或直接调控沙门菌T6SS核心组分的转录及表达。沙门菌中T6SS系统的调控有待进一步研究。
[Abstract]:Salmonella is an important zoonotic pathogen, which is widely distributed in nature. Most Salmonella have pathogenicity to human and animal, and can cause many different clinical manifestations of Salmonella. Salmonella is divided into intestinal Salmonella and Bongo Salmonella species. Salmonella serotype has more than 2500 serotypes, among which Salmonella enteritidis and Salmonella typhimurium are particularly important in human foodborne salmonella. Type 6 secretion system,T6SS is a new protein transporter system found in Gram-negative bacteria in recent years. It can transport protein to extracellular. T6SS is widely found in bacteria. Its structure consists of Icm F family, ATP enzyme-Clp VN Fha regulatory protein, and secretory Vgr G and Hcp proteins. At present, it has been found that T6SS plays an important role in the adaptability of bacteria to the environment, the pathogenicity of host cells and the formation of biofilm. Like other secretory systems, T6SS transcription and expression levels are tightly regulated. In Vibrio cholerae, the density sensing (Quorum sensing,QS) system lux S gene can significantly affect the transcription level of the main components of T6SS in Vibrio cholerae. Therefore, the effect of QS on T6SS in Salmonella was studied by constructing Salmonella lux S gene deletion strain. 1. Establishment and Application of multiple PCR Assay for the Detection of Salmonella virulence Factor the rapid and sensitive detection of Salmonella virulence gene was carried out by establishing multiple PCR method for different virulence island genes of Salmonella. According to the Gen Bank sequence, 16 pairs of primers were designed and synthesized, the reaction conditions were optimized, and four groups of multiplex PCR methods were established. The virulence genes of 124 Salmonella strains were detected by using the multiplex PCR method. The results showed that the distribution rate of the other 15 virulence genes was more than 90%, except that the sop E gene distribution rate was 15%. The pathogenicity of the representative strains to mice was determined. The results showed that salmonella could lead to morbidity and death of mice. The results of drug sensitivity test showed that the resistance rate of Salmonella to erythromycin, azithromycin, clindamycin, neomycin, spectinomycin, tetracycline and rifampicin was more than 90%. Construction and Biological characteristics of Salmonella lux S Gene deletion strain; Red homologous recombination system was used to construct Salmonella lux S gene deletion strain and its growth rate, motility and biofilm formation were studied. The pathogenicity and RAW264.7 adhesion invasion of mouse macrophages were studied. The results showed that, Lux S had no significant effect on the growth rate, biofilm formation, pathogenicity and cell adhesion and invasion of Salmonella. But the motility of lux S deletion strain was significantly higher than that of wild strain. Analysis of the effect of Lux S protein on T6SS by density Induction system; cloning and purification of Hcp1,Hcp2 and Clp V proteins, the core components of T6SS, to prepare the corresponding immune serum. The transcription and protein expression of hcp1,hcp2 and clp V in lux S gene deletion strains were detected by fluorescence quantitative PCR and Western blotting. The results showed that there was no significant difference in the transcription and expression levels of the core components of T6SS between the deletion strains of, lux S gene and wild strains in logarithmic growth period and platform stage. This study showed that QS Lux S protein did not affect or directly regulate the transcription and expression of the core components of Salmonella T6SS. The regulation of T6SS system in Salmonella needs further study.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：2269609
[Abstract]:Salmonella is an important zoonotic pathogen, which is widely distributed in nature. Most Salmonella have pathogenicity to human and animal, and can cause many different clinical manifestations of Salmonella. Salmonella is divided into intestinal Salmonella and Bongo Salmonella species. Salmonella serotype has more than 2500 serotypes, among which Salmonella enteritidis and Salmonella typhimurium are particularly important in human foodborne salmonella. Type 6 secretion system,T6SS is a new protein transporter system found in Gram-negative bacteria in recent years. It can transport protein to extracellular. T6SS is widely found in bacteria. Its structure consists of Icm F family, ATP enzyme-Clp VN Fha regulatory protein, and secretory Vgr G and Hcp proteins. At present, it has been found that T6SS plays an important role in the adaptability of bacteria to the environment, the pathogenicity of host cells and the formation of biofilm. Like other secretory systems, T6SS transcription and expression levels are tightly regulated. In Vibrio cholerae, the density sensing (Quorum sensing,QS) system lux S gene can significantly affect the transcription level of the main components of T6SS in Vibrio cholerae. Therefore, the effect of QS on T6SS in Salmonella was studied by constructing Salmonella lux S gene deletion strain. 1. Establishment and Application of multiple PCR Assay for the Detection of Salmonella virulence Factor the rapid and sensitive detection of Salmonella virulence gene was carried out by establishing multiple PCR method for different virulence island genes of Salmonella. According to the Gen Bank sequence, 16 pairs of primers were designed and synthesized, the reaction conditions were optimized, and four groups of multiplex PCR methods were established. The virulence genes of 124 Salmonella strains were detected by using the multiplex PCR method. The results showed that the distribution rate of the other 15 virulence genes was more than 90%, except that the sop E gene distribution rate was 15%. The pathogenicity of the representative strains to mice was determined. The results showed that salmonella could lead to morbidity and death of mice. The results of drug sensitivity test showed that the resistance rate of Salmonella to erythromycin, azithromycin, clindamycin, neomycin, spectinomycin, tetracycline and rifampicin was more than 90%. Construction and Biological characteristics of Salmonella lux S Gene deletion strain; Red homologous recombination system was used to construct Salmonella lux S gene deletion strain and its growth rate, motility and biofilm formation were studied. The pathogenicity and RAW264.7 adhesion invasion of mouse macrophages were studied. The results showed that, Lux S had no significant effect on the growth rate, biofilm formation, pathogenicity and cell adhesion and invasion of Salmonella. But the motility of lux S deletion strain was significantly higher than that of wild strain. Analysis of the effect of Lux S protein on T6SS by density Induction system; cloning and purification of Hcp1,Hcp2 and Clp V proteins, the core components of T6SS, to prepare the corresponding immune serum. The transcription and protein expression of hcp1,hcp2 and clp V in lux S gene deletion strains were detected by fluorescence quantitative PCR and Western blotting. The results showed that there was no significant difference in the transcription and expression levels of the core components of T6SS between the deletion strains of, lux S gene and wild strains in logarithmic growth period and platform stage. This study showed that QS Lux S protein did not affect or directly regulate the transcription and expression of the core components of Salmonella T6SS. The regulation of T6SS system in Salmonella needs further study.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
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相关期刊论文 前2条
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2 曹恬雪;蒋文灿;何文成;纪凤仙;魏志刚;;沙门氏菌毒力因子的研究进展[J];中国预防兽医学报;2014年04期
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