阪崎克罗诺肠杆菌类脂A磷酸乙醇胺转移酶相关基因的鉴定

发布时间：2018-04-18 09:10

本文选题：阪崎克罗诺肠杆菌 + 脂多糖　；参考：《江南大学》2017年硕士论文

【摘要】：阪崎克罗诺肠杆菌(Cronobacter sakazakii)是一种食源性革兰氏阴性条件致病菌,它主要污染婴儿配方奶粉,会引起菌血症、新生儿脑膜炎、败血症及坏死性小肠炎等多种疾病。类脂A分子位于革兰氏阴性细菌的外膜外层,对于细胞的稳定性、渗透性及细菌的致病性起着重要的作用,且其分子结构与细菌致病能力有关。在大肠杆菌(Escherichia coli)和沙门氏菌(Salmonella typhimurium)中存在eptA基因,它编码的磷酸乙醇胺转移酶可以修饰类脂A分子结构。本论文利用基因表达和染色体基因敲除技术,构建一系列Cronobacter sakazakii BAA894基因工程菌,实验确定了ESA_RS09200为C.sakazakii BAA894中编码磷酸乙醇胺转移酶的基因,并且对该基因在C.sakazakii BAA894致病能力中所起作用进行探究。本课题的主要研究结论如下:(1)实验发现C.sakazakii BAA894在低pH(pH 5.0)条件下,类脂A结构上发生磷酸乙醇胺的修饰。通过基因组同源比对,发现BAA894中两个基因ESA_RS09200和ESA_RS16425与E.coli和S.typhimurium中eptA基因的同源性很高。(2)将ESA_RS09200和ESA_RS16425在E.coli W3110和C.sakazakii BAA894中过量表达,在pH 5.0条件下生长,提取类脂A并进行结构鉴定,证明ESA_RS09200编码磷酸乙醇胺氨基转移酶,而ESA_RS16425编码的酶不能修饰类脂A。(3)在C.sakazakii BAA894中分别敲除ESA_RS09200和ESA_RS16425,构建了对应的突变菌株WLL001和WLL002。在pH 5.0条件下生长,提取类脂A并进行结构鉴定,进一步证明ESA_RS09200编码磷酸乙醇胺氨基转移酶,而ESA_RS16425编码的酶不能修饰类脂A。测定了WLL001突变株的一系列生物学特性,包括生长曲线、细胞表面疏水性、外膜渗透性及抗生素最小抑菌浓度,并与野生型菌株进行对比,研究外膜LPS结构改变后,菌株的生化表型情况。(4)通过构建BAA894/pWSK29-lpxE、WLL001/pWSK29-lpxE、WLL003/pWSK29-lpxE和WLL003/pWSK29-lpxF菌株,使类脂A分子1位上或4’位上的磷酸基团缺失,证明ESA_RS09200编码磷酸乙醇胺氨基转移酶只能对4’位的磷酸进行修饰。(5)通过WLL001突变株活菌体刺激HEK-Blue hTLR4细胞和提取纯化突变株的脂多糖分子来刺激小鼠RAW264.7细胞,分析了类脂A结构改变对免疫识别的影响。通过细胞侵染实验发现WLL001突变株对Caco-2细胞的侵染能力下降,在THP-1巨噬细胞内的存活率降低。
[Abstract]:Cronobacter sakazakii) is a foodborne gram-negative conditional pathogen, which mainly pollutes infant formula and can cause many diseases such as bacteremia, neonatal meningitis, septicemia and necrotizing enteritis.Lipoid A molecules are located in the outer layer of the outer membrane of Gram-negative bacteria and play an important role in cell stability, permeability and pathogenicity of bacteria, and their molecular structure is related to the pathogenicity of bacteria.The eptA gene is present in Escherichia coli and Salmonella typhimurium, which encodes a phosphoethanolamine transferase that modifies the molecular structure of lipids A.In this paper, a series of Cronobacter sakazakii BAA894 genetically engineered bacteria were constructed by using gene expression and chromosome knockout techniques. ESA_RS09200 was identified as the gene encoding ethanolamine phosphotransferase in C.sakazakii BAA894.The role of the gene in the pathogenicity of C.sakazakii BAA894 was investigated.The main conclusions of this study are as follows: 1) the experimental results show that the modification of ethanolamine phosphate on the structure of lipids A occurs in C.sakazakii BAA894 under the condition of low pH(pH 5.0).It was found that ESA_RS09200 and ESA_RS16425 in BAA894 were highly homologous to eptA genes in E.coli and S.typhimurium.) ESA_RS09200 and ESA_RS16425 were overexpressed in E.coli W3110 and C.sakazakii BAA894. They grew at pH 5.0. Lipoid A was extracted and identified.It was proved that ESA_RS09200 encodes ethanolamine aminotransferase, while the enzyme encoded by ESA_RS16425 can not modify lipids A. (3) ESA_RS09200 and ESARS16425 were knocked out in C.sakazakii BAA894, respectively. The corresponding mutant strains WLL001 and WLL002 were constructed.Under the condition of pH 5.0, lipids A were extracted and identified. It was further proved that ESA_RS09200 encodes ethanolamine aminotransferase, while the enzyme encoded by ESA_RS16425 can not modify lipids.A series of biological characteristics of WLL001 mutants, including growth curve, hydrophobicity of cell surface, permeability of outer membrane and minimal inhibitory concentration of antibiotics, were determined and compared with wild-type strains. The changes of outer membrane LPS structure were studied.By constructing BAA894 / pWSK29-lpxE WLL001 / pWSK29-lpxE and WLL003/pWSK29-lpxF strain WLL003 / pWSK29-lpxE and WLL003/pWSK29-lpxF, the phosphoric acid group at one or four 'position of lipoid A molecule was deleted.It was proved that ESA_RS09200 encoded ethanolamine aminotransferase could only modify 4 '-position phosphoric acid. (5) HEK-Blue hTLR4 cells were stimulated by live WLL001 mutant and lipopolysaccharide molecules of purified mutant were extracted to stimulate RAW264.7 cells in mice.The effect of structural changes of lipoid A on immune recognition was analyzed.The ability of WLL001 mutant to infect Caco-2 cells was decreased and the survival rate in THP-1 macrophages was decreased.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;R378

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