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杀鱼爱德华氏菌硫胺素转运基因的毒力功能研究
刘昕
学位类型硕士
导师孙黎
2022-05-18
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
关键词杀鱼爱德华氏菌 硫胺素 转运蛋白 环二聚鸟苷单磷酸 毒力
摘要

硫胺素(维生素 B1)是焦磷酸硫胺素(Thiamine pyrophosphateTPP)的前体,是几乎所有生物必需的营养物质。TPP 参与细胞内多种代谢过程,包括糖类、脂质和氨基酸代谢,但其在细菌致病性中的作用尚不清楚。细菌通过自身合成或转运外源硫胺素获得 TPP。环二聚鸟苷单磷酸(c-di-GMP)是细菌的主要第二信使分子,参与调节包括感染在内的多种生物学过程。TPP 可能在c-di-GMP 信号转导中发挥作用,因为它是核糖类生物合成的必需辅酶,而核糖是 c-di-GMP 成的前体。然而,这种可能性尚无实验证据佐证。杀鱼爱德华氏菌(Edwardsiella piscicida)是一种重要的鱼类致病菌,具有广泛的宿主范围。TPP c-di-GMP 杀鱼爱德华氏菌致病性中的作用有待解析。在本研究中,我们鉴定了一个杀鱼爱德华氏菌硫胺素转运蛋白(Thiamine TransporterTT)操纵子,该操纵子由 thiBthiP thiQ 组成,编码一种ThiBPQ 型硫胺素转运系统。敲除 TT 操纵子导致细胞内 TPP 水平显著降低,这表明杀鱼爱德华氏菌可能主要通过摄取硫胺素来获TPPTPP 含量降低导致杀鱼爱德华氏菌对大菱鲆的致病性下降,细菌运动能力和宿主细胞黏附能力显著降低,鞭毛和黏附素基因表达显著降低,这表明硫胺素参与了杀鱼爱德华氏菌毒力调控。此外,TPP 含量降低导致细菌胞内 c-di-GMP含量显著下降,而在 TT 突变株中引入外源 c-di-GMP 合成酶则使杀鱼爱德华氏菌恢复了毒力基因的表达、运动能力和宿主细胞黏附能力。综上所述,本研究揭示了硫胺素摄取和 c-di-GMP 在杀鱼爱德华氏菌致病性中的重要作用,为杀鱼爱德华氏菌的防治提供了新的视角。

其他摘要
Thiamine (Vitamin B1) is the precursor of thiamine pyrophosphate (TPP), an essential nutrient for almost all organisms. TPP is involved in a variety of intracellular metabolic processes, including carbohydrate, lipid and amino acid metabolism, but the mechanism of its role in bacterial pathogenicity remains unclear. Bacteria acquire TPP
by biosynthesis or by transportation of exogenous thiamine. Bis - (3’ - 5’)- cyclic dimeric guanosine monophosphate (c-di-GMP) is a major second messenger of bacteria and regulates a variety of biological processes including infection. TPP might have a role in c-di-GMP signaling, as it functions as an essential coenzyme for the biosynthesis
of ribose, the precursor of c-di-GMP. However, this mechanism has not been supported yet by solid experimental evidence. The roles of TPP and c-di-GMP in the pathogenicity of Edwardsiella piscicida, a severe fish pathogen with a broad host range, are to be discovered. In this study, we characterized a thiBPQ type of thiamine transporter (TT)
operon in E. piscicida, which is composed of thiB, thiP and thiQ. Deletion of the TT operon resulted in significantly reduced intracellular TPP level, indicating that E. piscicida likely acquires TPP mainly through thiamine uptake. The TPP starvation in E. piscicida led to attenuated overall pathogenicity to host Scophthalmus maximus,
markedly impaired abilities associated with motility and host cell adhesion, as well as decreased expression of certain flagellar and adhesion genes, indicating the involvement of thiamine in the virulence regulation of E. piscicida. Moreover, TPP suppression decreased the c-di-GMP level, and introducing into the TPP-suppressed mutant strain an exogenous diguanylate cyclase for c-di-GMP synthesis restored the
virulence gene expression and the capacities for motility and host cell adhesion. Taken together, this work reveals the importance of thiamine uptake and c-di-GMP in the pathogenicity of E. piscicida, which may provide a new research strategy for the control of E. piscicida.
学科门类工学 ; 工学::生物工程
资助项目Taishan Scholar Program of Shandong Province ; National Key R&D Program of China[2018YFD0900500]
语种中文
目录
第一章 绪论.............................................................................................1
1.1 杀鱼爱德华氏菌...............................................................................................1
1.1.1 杀鱼爱德华氏菌概述................................................................................1
1.1.2 杀鱼爱德华氏菌感染过程........................................................................1
1.1.3 杀鱼爱德华氏菌主要毒力因子................................................................2
1.1.4 鱼类爱德华氏菌病的防治........................................................................6
1.2 大菱鲆...............................................................................................................7
1.3 硫胺素...............................................................................................................8
1.3.1 细菌中的硫胺素........................................................................................8
1.3.2 硫胺素对细菌毒力的影响........................................................................9
1.4 c-di-GMP ........................................................................................................10
1.5 本研究的目的和意义.....................................................................................11
第二章 材料和方法 ..............................................................................13
2.1 材料.................................................................................................................13
2.1.1 培养基及抗生素......................................................................................13
2.1.2 实验菌株..................................................................................................13
2.1.3 实验引物..................................................................................................15
2.1.4 实验动物..................................................................................................19
2.1.5 实验试剂..................................................................................................19
2.1.6 实验仪器..................................................................................................21
2.2 方法.................................................................................................................22
2.2.1 生物信息学和序列分析方法..................................................................22
2.2.2 TT 缺失突变株的构建............................................................................22
2.2.3 回补株及过表达菌株构建......................................................................26
2.2.4 报告基因质粒构建..................................................................................29
2.2.5 生长曲线测定..........................................................................................30
2.2.6 体内感染实验..........................................................................................30
2.2.7 大菱鲆外周血淋巴细胞炎症因子转录水平检测..................................31
2.2.8 细菌运动性实验......................................................................................33
2.2.9 鞭毛基因和黏附基因表达检测..............................................................33
2.2.10 PBLs 细胞黏附实验..............................................................................34
2.2.11 荧光强度定量分析................................................................................34
2.2.12 统计分析................................................................................................35
第三章 实验结果与分析 ......................................................................37
3.1 ThiBPQ 蛋白序列比对结果分析 ..................................................................37
3.2 TT 突变株和回补株构建...............................................................................39
3.2.1 TT 上下游同源臂扩增产物及 overlap-PCR..........................................39
3.2.2 同源重组..................................................................................................39
3.2.3 TT 突变株验证........................................................................................40
3.2.4 回补株构建..............................................................................................41
3.3 TT 敲除对杀鱼爱德华氏菌胞内 TPP 的影响..............................................41
3.4 TT 敲除对杀鱼爱德华氏菌生长的影响.......................................................42
3.5 TT 敲除对杀鱼爱德华氏菌毒力的影响.......................................................43
3.6 TT 敲除对大菱鲆外周血淋巴细胞炎症因子表达的影响...........................44
3.7 TT 敲除对杀鱼爱德华氏菌运动能力和鞭毛基因表达的影响...................45
3.7.1 TT 敲除对杀鱼爱德华氏菌运动性的影响............................................45
3.7.2 TT 敲除对鞭毛基因表达的影响............................................................46
3.8 TT 敲除对杀鱼爱德华氏菌黏附宿主细胞能力和黏附基因表达的影响...47
3.8.1 TT 敲除对杀鱼爱德华氏菌黏附宿主细胞能力的影响........................47
3.8.2 TT 敲除对杀鱼爱德华氏菌黏附基因表达的影响................................48
3.8.3 ETAE_1902 ETAE_2842 过表达对 ZW1ΔTT 黏附能力的影响 ....49
3.9 c-di-GMP 参与硫胺素摄取对杀鱼爱德华氏菌毒力调控 ...........................50
3.9.1 TT 敲除对胞内 c-di-GMP 相对含量的影响 .........................................50
3.9.2 过表达 adrA 对杀鱼爱德华氏菌鞭毛和黏附基因表达的影响............51
3.9.3 过表达 adrA 对杀鱼爱德华氏菌运动性的影响....................................52
3.9.4 过表达 adrA 对杀鱼爱德华氏菌黏附 PBLs 细胞能力的影响.............53
第四章 讨论...........................................................................................55
第五章 结论与展望 ..............................................................................61
参考文献...................................................................................................63
.......................................................................................................75
作者简历及攻读学位期间发表的学术论文与研究成果......................77
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/178300
专题中国科学院海洋研究所
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刘昕. 杀鱼爱德华氏菌硫胺素转运基因的毒力功能研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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