阪崎克罗诺杆菌LPS结构基因waaC的鉴定及其对菌膜形成影响研究
发布时间：2019-02-13 01:13
【摘要】：阪崎克罗诺杆菌是一种自然界中广泛存在的,主要污染婴儿配方奶粉的食源性革兰氏阴性条件致病菌。由于其明确的致病性,阪崎克罗诺杆菌被列为A类致病菌。它可以引起新生儿脑膜炎、败血病、菌血症及坏死性小肠结膜炎等疾病,死亡率可高达80%。然而,阪崎克罗诺杆菌的致病机制还不清楚。阪崎克罗诺杆菌可能通过在肠道内增殖并粘附及入侵肠上皮细胞而致病,而菌膜的形成可能在这个过程中起着重要作用。脂多糖是阪崎克罗诺杆菌细胞表面的主要致病性大分子,与大肠杆菌、沙门氏菌和空肠弯曲菌等一些革兰氏阴性菌的菌膜形成密切相关,但是阪崎克罗诺杆菌的脂多糖生物合成途径中的多数基因及其与菌膜形成的关系尚未确定。 本课题对阪崎克罗诺杆菌的脂多糖生物合成途径关键基因waaC进行了鉴定,并从多方面研究该基因在菌膜形成中的重要作用,从而探索阪崎克罗诺杆菌的脂多糖结构与菌膜形成之间的关系。主要结论如下: (1)证实了ESA_04107为阪崎克罗诺杆菌的脂多糖生物合成途径中的庚糖基转移酶I的编码基因waaC。首先,通过同源比对发现阪崎克罗诺杆菌BAA-894中ESA_04107的编码序列与大肠杆菌W3110的WaaC同源性最高;其次,通过敲除ESA_04107构建了阪崎克罗诺杆菌突变株LWW02;最后,从LWW02中提取脂多糖,用SDS-聚丙烯酰胺凝胶电泳和薄层层析进行结构分析,用DEAE-纤维素阴离子交换层析纯化,并用ESI/MS确定其分子结构。 (2)研究阪崎克罗诺杆菌脂多糖结构突变株LWW02的菌膜形成能力。与野生型BAA-894相比,LWW02的生长受到影响,但是在亲疏水介质表面的菌膜形成能力却增强,表明阪崎克罗诺杆菌的脂多糖结构与菌膜形成能力密切相关。 (3)探究阪崎克罗诺杆菌脂多糖结构对细胞其他特性的影响。与野生型BAA-894相比,LWW02的细胞表面疏水性、外膜通透性、自凝集性以及胞外物分泌量等都有了显著提高。这些因素可能是导致LWW02菌膜形成能力增强的主要原因。本课题在鉴定阪崎克罗诺杆菌的脂多糖生物合成途径关键基因waaC的基础上,将阪崎克罗诺杆菌的外膜结构组分脂多糖与菌膜形成联系起来,从多角度探究了阪崎克罗诺杆菌脂多糖影响菌膜形成能力变化的机制,对探究阪崎克罗诺杆菌的致病机理,检测与控制阪崎克罗诺杆菌等都具有重要意义。
[Abstract]:Hanshizaki Crosenia is a foodborne gram-negative opportunistic pathogen that is widely found in nature and mainly contaminates infant formula. Because of its definite pathogenicity, Hanshizaki Crosenia is classified as a class A pathogen. It can cause diseases such as neonatal meningitis, septicaemia, bacteremia and necrotizing small bowel conjunctivitis. However, the pathogenetic mechanism of Hanshizaki Croix is unclear. Hanshizaki Crosenia may cause disease by proliferation, adhesion and invasion of intestinal epithelial cells, and the formation of bacterial membrane may play an important role in this process. Lipopolysaccharide is the major pathogenicity macromolecule on the cell surface of Hanshizaki Crosenia, which is closely related to the membrane formation of some gram-negative bacteria such as Escherichia coli, Salmonella and Campylobacter jejuni. However, most of the genes in the lipopolysaccharide biosynthesis pathway of Hanshizaki Crosenia and their relationship with membrane formation have not been determined. In this paper, the key gene waaC of lipopolysaccharide biosynthesis pathway of Hanshizaki Crosenia was identified, and the important role of this gene in membrane formation was studied from many aspects. To explore the relationship between lipopolysaccharide structure and membrane formation of Hanshizaki Crosenia. The main conclusions are as follows: (1) it is confirmed that ESA_04107 is the encoding gene waaC. of heptose transferase I in the lipopolysaccharide biosynthesis pathway of Hanshizaki. Firstly, the coding sequence of ESA_04107 in BAA-894 of Hanshizaki Crosenia was found to have the highest homology with the WaaC of Escherichia coli W3110. Secondly, the mutant LWW02; was constructed by knockout ESA_04107. Finally, the lipopolysaccharide was extracted from LWW02. The structure was analyzed by SDS- polyacrylamide gel electrophoresis and thin-layer chromatography. The structure was purified by DEAE- cellulose anion exchange chromatography, and its molecular structure was determined by ESI/MS. (2) to study the membrane forming ability of LWW02. Compared with wild-type BAA-894, the growth of LWW02 was affected, but the membrane formation ability on the surface of hydrophilic medium was enhanced, indicating that the lipopolysaccharide structure of Hanshizaki Croix was closely related to the membrane formation ability. (3) to investigate the effect of lipopolysaccharide structure of Hanshin Crosaki bacillus on other characteristics of cells. Compared with wild-type BAA-894, the surface hydrophobicity, outer membrane permeability, self-agglutination and extracellular secretion of LWW02 were significantly increased. These factors may be the main reasons for the enhancement of membrane formation ability of LWW02 bacteria. On the basis of identifying the key gene of lipopolysaccharide biosynthesis pathway of Hanshizaki, waaC, the lipopolysaccharide of the outer membrane structure of Hanshizaki Crosenia was associated with the formation of the membrane. In this paper, the mechanism of lipopolysaccharide (LPS) from Hanshazaki Crosenia on the change of membrane formation ability is studied, which is of great significance for the study of pathogenicity, detection and control of Hanshizaki Crosenia.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
[Abstract]:Hanshizaki Crosenia is a foodborne gram-negative opportunistic pathogen that is widely found in nature and mainly contaminates infant formula. Because of its definite pathogenicity, Hanshizaki Crosenia is classified as a class A pathogen. It can cause diseases such as neonatal meningitis, septicaemia, bacteremia and necrotizing small bowel conjunctivitis. However, the pathogenetic mechanism of Hanshizaki Croix is unclear. Hanshizaki Crosenia may cause disease by proliferation, adhesion and invasion of intestinal epithelial cells, and the formation of bacterial membrane may play an important role in this process. Lipopolysaccharide is the major pathogenicity macromolecule on the cell surface of Hanshizaki Crosenia, which is closely related to the membrane formation of some gram-negative bacteria such as Escherichia coli, Salmonella and Campylobacter jejuni. However, most of the genes in the lipopolysaccharide biosynthesis pathway of Hanshizaki Crosenia and their relationship with membrane formation have not been determined. In this paper, the key gene waaC of lipopolysaccharide biosynthesis pathway of Hanshizaki Crosenia was identified, and the important role of this gene in membrane formation was studied from many aspects. To explore the relationship between lipopolysaccharide structure and membrane formation of Hanshizaki Crosenia. The main conclusions are as follows: (1) it is confirmed that ESA_04107 is the encoding gene waaC. of heptose transferase I in the lipopolysaccharide biosynthesis pathway of Hanshizaki. Firstly, the coding sequence of ESA_04107 in BAA-894 of Hanshizaki Crosenia was found to have the highest homology with the WaaC of Escherichia coli W3110. Secondly, the mutant LWW02; was constructed by knockout ESA_04107. Finally, the lipopolysaccharide was extracted from LWW02. The structure was analyzed by SDS- polyacrylamide gel electrophoresis and thin-layer chromatography. The structure was purified by DEAE- cellulose anion exchange chromatography, and its molecular structure was determined by ESI/MS. (2) to study the membrane forming ability of LWW02. Compared with wild-type BAA-894, the growth of LWW02 was affected, but the membrane formation ability on the surface of hydrophilic medium was enhanced, indicating that the lipopolysaccharide structure of Hanshizaki Croix was closely related to the membrane formation ability. (3) to investigate the effect of lipopolysaccharide structure of Hanshin Crosaki bacillus on other characteristics of cells. Compared with wild-type BAA-894, the surface hydrophobicity, outer membrane permeability, self-agglutination and extracellular secretion of LWW02 were significantly increased. These factors may be the main reasons for the enhancement of membrane formation ability of LWW02 bacteria. On the basis of identifying the key gene of lipopolysaccharide biosynthesis pathway of Hanshizaki, waaC, the lipopolysaccharide of the outer membrane structure of Hanshizaki Crosenia was associated with the formation of the membrane. In this paper, the mechanism of lipopolysaccharide (LPS) from Hanshazaki Crosenia on the change of membrane formation ability is studied, which is of great significance for the study of pathogenicity, detection and control of Hanshizaki Crosenia.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378
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