本文選題：阪崎克羅諾腸桿菌 + 脂多糖　； 參考：《江南大學》2017年碩士論文

【摘要】：阪崎克羅諾腸桿菌(Cronobacter sakazakii)是一種食源性革蘭氏陰性條件致病菌,它主要污染嬰兒配方奶粉,會引起菌血癥、新生兒腦膜炎、敗血癥及壞死性小腸炎等多種疾病。類脂A分子位于革蘭氏陰性細菌的外膜外層,對于細胞的穩(wěn)定性、滲透性及細菌的致病性起著重要的作用,且其分子結構與細菌致病能力有關。在大腸桿菌(Escherichia coli)和沙門氏菌(Salmonella typhimurium)中存在eptA基因,它編碼的磷酸乙醇胺轉移酶可以修飾類脂A分子結構。本論文利用基因表達和染色體基因敲除技術,構建一系列Cronobacter sakazakii BAA894基因工程菌,實驗確定了ESA_RS09200為C.sakazakii BAA894中編碼磷酸乙醇胺轉移酶的基因,并且對該基因在C.sakazakii BAA894致病能力中所起作用進行探究。本課題的主要研究結論如下:(1)實驗發(fā)現C.sakazakii BAA894在低pH(pH 5.0)條件下,類脂A結構上發(fā)生磷酸乙醇胺的修飾。通過基因組同源比對,發(fā)現BAA894中兩個基因ESA_RS09200和ESA_RS16425與E.coli和S.typhimurium中eptA基因的同源性很高。(2)將ESA_RS09200和ESA_RS16425在E.coli W3110和C.sakazakii BAA894中過量表達,在pH 5.0條件下生長,提取類脂A并進行結構鑒定,證明ESA_RS09200編碼磷酸乙醇胺氨基轉移酶,而ESA_RS16425編碼的酶不能修飾類脂A。(3)在C.sakazakii BAA894中分別敲除ESA_RS09200和ESA_RS16425,構建了對應的突變菌株WLL001和WLL002。在pH 5.0條件下生長,提取類脂A并進行結構鑒定,進一步證明ESA_RS09200編碼磷酸乙醇胺氨基轉移酶,而ESA_RS16425編碼的酶不能修飾類脂A。測定了WLL001突變株的一系列生物學特性,包括生長曲線、細胞表面疏水性、外膜滲透性及抗生素最小抑菌濃度,并與野生型菌株進行對比,研究外膜LPS結構改變后,菌株的生化表型情況。(4)通過構建BAA894/pWSK29-lpxE、WLL001/pWSK29-lpxE、WLL003/pWSK29-lpxE和WLL003/pWSK29-lpxF菌株,使類脂A分子1位上或4’位上的磷酸基團缺失,證明ESA_RS09200編碼磷酸乙醇胺氨基轉移酶只能對4’位的磷酸進行修飾。(5)通過WLL001突變株活菌體刺激HEK-Blue hTLR4細胞和提取純化突變株的脂多糖分子來刺激小鼠RAW264.7細胞,分析了類脂A結構改變對免疫識別的影響。通過細胞侵染實驗發(fā)現WLL001突變株對Caco-2細胞的侵染能力下降,在THP-1巨噬細胞內的存活率降低。
[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.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78;R378

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