实验海洋生物学重点实验室知识库

迟缓爱德华氏菌（Edwardsiella tarda）是致病性革兰氏阴性菌，可感染包括哺乳类、爬行动物和鱼类等在内的多种宿主。E. tarda的一个很重要的毒力特征是能够在宿主巨噬细胞中存活并增殖，但相关的分子机制仍有待深入研究。在本研究中，我们建立了E. tarda感染小鼠巨噬细胞RAW264.7的细胞感染模型，通过转录组测序技术分析了E. tarda在感染不同时间点诱导的宿主细胞全基因表达谱。为此目的，RAW264.7细胞分别用E. tarda活菌和E. tarda灭活菌处理4 h和8 h，随后检测两组RAW264.7细胞的基因转录。结果表明，与E. tarda活菌处理相比，E. tarda灭活菌处理引起RAW264.7细胞更强烈的应答反应，且差异表达基因显著富集在与免疫相关的GO条目和KEGG通路中。与E. tarda灭活菌处理相比，E. tarda活菌显著抑制了大量免疫相关基因的表达，包括RIG-I样受体、细胞因子、干扰素相关基因等，且一些免疫相关基因之间形成复杂的相互作用网络。进一步研究发现，在E. tarda活菌感染的细胞中，iNOS的表达量和NO的产生量都显著低于灭活菌处理的细胞。Western blot结果表明，随着感染时间的延长，灭活菌处理的细胞的NF-κB p65的磷酸化水平不断升高，但活菌感染组的磷酸化水平未明显升高，且在各个时间点都低于灭活菌处理组，暗示着E. tarda可能通过调控宿主NF-κB通路激活及下游杀菌效应分子的生成来促进其在胞内的存活和增殖。

接着，我们建立了E. tarda感染BALB/c小鼠的体内感染模型，通过抗体芯片技术检测了E. tarda感染后小鼠血浆中96个细胞因子的表达情况。与PBS处理的对照组相比，E. tarda感染组在感染后6 h和24 h，分别呈现出31个和26个差异表达的细胞因子。这些差异表达细胞因子显著富集在与细胞迁移和趋化因子响应相关的GO条目中以及与免疫、疾病和感染相关的KEGG通路中，并且IL-6和TNF-α等差异表达细胞因子之间形成复杂的蛋白互作网络。进一步研究表明，利用小鼠IL-6重组蛋白预处理RAW264.7细胞显著抑制E. tarda在胞内的感染，而干扰IL-6的表达可显著增强E. tarda在胞内的增殖。在E. tarda感染过程中，干扰细胞IL-6表达显著降低TNF-α、iNOS和IL-27的表达以及NO的产生。利用小鼠TNF-α重组蛋白预处理RAW264.7细胞也会显著抑制E. tarda在胞内的感染。我们进而检测了牙鲆（Paralichthys olivaceus）TNF-α在E. tarda感染中的作用，结果显示，干扰牙鲆TNF-α的表达可显著增强E. tarda在牙鲆鳃细胞FG-9307内的增殖，暗示着E. tarda感染引起的小鼠和鱼类的免疫反应可能存在一定的相似性。

趋化因子是一组在细胞迁移、炎症和免疫防御中发挥重要作用的细胞因子。我们在上述E. tarda感染小鼠的实验中鉴定到了差异表达的细胞因子，其中有一系列趋化因子，包括CXCL10。因此，我们探究了鱼类CXCL10是否和哺乳动物CXCL10一样参与宿主抗菌免疫反应。我们从牙鲆中鉴定并克隆了趋化因子CXCL10（命名为PoCXCL10）并对其功能进行了研究。PoCXCL10在结构上含有一个自由延伸的N端、三个β片层和C端的α螺旋。正常生理条件下，PoCXCL10在多个组织中有不同水平的表达。E. tarda感染后，肝脏、脾脏和头肾中PoCXCL10的表达显著上调。重组rPoCXCL10蛋白（rPoCXCL10）能够以剂量依赖的方式在体外诱导牙鲆外周血白细胞（PBLs）迁移，并促进细胞因子IL-6、CXCL8、IL-1β、TNF-α和IL-10的表达。rPoCXCL10能够结合革兰氏阳性菌和革兰氏阴性菌，并抑制E. tarda、海豚链球菌Streptococcus iniae、鳗弧菌Vibrio anguillarum和哈维氏弧菌Vibrio harveyi的生长。此外，rPoCXCL10对V. anguillarum和V. harveyi具有直接的杀菌活性。为了研究PoCXCL10功能的结构依赖性，我们构建了一个缺少C端α-螺旋区域的PoCXCL10突变体蛋白，rPoCXCL10M。实验结果表明，rPoCXCL10M和rPoCXCL10都能结合牙鲆PBLs，且rPoCXCL10M的趋化活性与rPoCXCL10相当。然而，rPoCXCL10M的杀菌活性显著低于rPoCXCL10，说明C端α-螺旋区域对体外杀菌活性至关重要。体内实验结果表明，rPoCXCL10和rPoCXCL10M可显著减少E. tarda和V. anguillarum在牙鲆组织中的传播和定殖。

综上所述，我们利用小鼠为感染模型，探究了E. tarda胞内感染的相关分子通路，确定了E. tarda诱导的血浆细胞因子的表达模式，为深入研究抗E. tarda感染免疫提供了细胞因子靶点。我们进一步将哺乳动物实验结果扩展到鱼类，发现趋化因子CXCL10在牙鲆抗E. tarda感染免疫中发挥重要作用，研究结果提升了对鱼类趋化因子免疫功能的理解，也为鱼类迟缓爱德华氏菌病的免疫防控提供了理论基础。

Edwardsiella tarda is a pathogenic Gram-negative bacterium that can infect a variety of hosts, including mammals, reptiles and fish. One of the most important virulence characteristics of E. tarda is its ability to survive and replicate in host macrophages. However, the related molecular mechanisms remain to be further studied. In this study, we analyzed the transcriptome profiles of RAW264.7 cells infected with live E. tarda or treated with dead E. tarda for 4 h and 8 h. The results showed that dead E. tarda induced a stronger response in RAW264.7 cells, and the differentially expressed genes were significantly enriched in immune-related GO terms and KEGG pathways. Compared with dead E. tarda-treated cells, live E. tarda-treated cells showed significant suppression in the induction of a large number of immune-related genes, including RIG-I-like receptors, cytokines, and interferon-related genes. Some of these immune-related genes formed complicated interaction networks with each other. The expression of iNOS and the production of nitric oxide (NO) in live E. tarda-treated cells was significantly lower than that in dead E. tarda-treated cells. The results of Western blot showed that in dead E. tarda-treated RAW264.7 cells, phosphorylation of NF-κB p65 increased with the prolongation of infection time. While in live E. tarda-treated cells, the phosphorylation of NF-κB p65 was apparently lower than that in dead E. tarda-treated cells at each time point, suggesting that E. tarda may contribute to its intracellular survival and replicate by regulating the activation of NF-κB pathway and the generation of bactericidal effector molecules.

Then, we established an in vivo model of E. tarda infected BALB/c mice and used the antibody array technology to identify the expression pattern of 96 cytokines in the plasma of E. tarda-infected mice. In total, 31 and 26 differentially expressed cytokines were identified at 6 h and 24 h post-infection, respectively. These differentially expressed cytokines were significantly enriched in GO terms related to cell migration and chemokine response as well as KEGG pathways related to immunity, disease and infection. Differentially expressed cytokines including IL-6 and TNF-α formed complicated interaction networks with each other. We found that pretreatment of RAW264.7 cells with mouse IL-6 recombinant protein inhibited the intracellular infection of E. tarda, while interference with IL-6 significantly enhanced the replicate of E. tarda. In addition, IL-6 knockdown markedly reduced the expression of TNF-α, iNOS, and IL-27 and the production of NO during E. tarda infection. TNF-α also showed an inhibitory effect on E. tarda infection, and interference with Japanese flounder Paralichthys olivaceus TNF-α promoted the replicate of E. tarda in flounder gill cells FG-9307, suggesting that there may exist correlation between the immune response of mice and that of fish induced by E. tarda infection.

Chemokines are a group of cytokines that play an important role in cell migration, inflammation, and immune defense. As said above, we have identified a number of differentially expressed chemokines, including CXCL10, in E. tarda-infected mice. We next examined whether fish CXCL10, like mammalian CXCL10, is involved in the host antimicrobial immune response. For this purpose, we investigated the expression and function of Japanese flounder CXCL10 (named PoCXCL10). Structurally, PoCXCL10 is composed of an extended N-terminal coil, three antiparallel β-strands, and a C-terminal α-helix. PoCXCL10 was expressed in multiple tissues at different levels under normal physiological conditions, and the expression was up-regulated in liver, spleen, and head kidney upon bacterial challenge. Recombinant PoCXCL10 (rPoCXCL10) induced the migration of Japanese flounder peripheral blood leukocytes (PBLs) in a dose-dependent manner. rPoCXCL10 also induced the expression of IL-6, TNF-α, CXCL8, IL-1β, and IL-10. rPoCXCL10 was able to bind both Gram-positive and Gram-negative bacteria, and inhibit the growth of E. tarda, Streptococcus iniae, Vibrio anguillarum, and Vibrio harveyi. Furthermore, rPoCXCL10 possessed direct bactericidal activity against V. anguillarum and V. harveyi. To examine whether the function of PoCXCL10 was structure dependent, we constructed a truncated rPoCXCL10, named rPoCXCL10M, which lacks the C-terminal α-helix region. Both rPoCXCL10M and rPoCXCL10 were able to bind PBLs and exhibited comparable chemotactic activities. However, the bactericidal activity of rPoCXCL10M was significantly lower than that of rPoCXCL10, indicating that the C-terminal α-helix region was essential for the bactericidal activity. In vivo experiments showed that rPoCXCL10 and rPoCXCL10M treatment could reduce the spread and colonization of E. tarda and V. anguillarum in flounder tissues.

In conclusion, we focused on the immune response of mice and fish to E. tarda infection. The molecular pathways for the intracellular infection of E. tarda were detected by high-throughput sequencing, and the expression pattern of cytokines in plasma of mice infected with E. tarda was detected by antibody microarray. Our results provided the potential plasma biomarkers for E. tarda infection and targets for the study of cytokines in host antimicrobial immunity. We applied these findings to the study of fish, and found that Japanese flounder CXCL10 played an important role in the immune defense against bacterial infection. These results add new insights into the immune mechanim of fish chemokines and provide a theoretical basis for the prevention and control of Edwardsiellasis.

第一章 引言... 1

1.1 迟缓爱德华氏菌... 1

1.1.1 迟缓爱德华氏菌概况... 1

1.1.2 迟缓爱德华氏菌致病机制研究... 2

1.1.3 迟缓爱德华氏菌免疫逃逸机制研究... 4

1.2 巨噬细胞... 5

1.2.1 巨噬细胞免疫功能概况... 5

1.2.2 巨噬细胞抗菌机制... 6

1.3 细胞因子... 8

1.3.1 细胞因子概况... 8

1.3.2 细胞因子受体及其信号通路... 8

1.3.3 细胞因子在宿主抗菌免疫中的功能研究... 10

1.4 趋化因子... 13

1.4.1 趋化因子概况... 13

1.4.2 趋化因子受体与信号转导机制... 14

1.4.3 趋化因子在宿主抗菌免疫中的功能研究... 15

1.4.4 硬骨鱼类趋化因子的功能研究... 16

1.5 本研究的目的与意义... 17

第二章 迟缓爱德华氏菌胞内感染及其诱导的细胞转录表达谱... 19

2.1 实验材料与方法... 19

2.1.1 实验细胞和菌株... 19

2.1.2 实验试剂与仪器... 19

2.1.3 迟缓爱德华氏菌的细胞感染模型建立... 21

2.1.4 细胞磷酸酶活性检测... 22

2.1.5 转录组测序分析... 23

2.1.6 蛋白相互作用网络分析... 23

2.1.7 实时荧光定量PCR（qRT-PCR）验证差异表达基因... 23

2.1.8 一氧化氮（NO）生成量检测... 24

2.1.9 NF-κB p65磷酸化水平检测... 25

2.2 实验结果... 25

2.2.1 E. tarda在RAW264.7细胞内的感染情况... 25

2.2.2 E. tarda感染对RAW264.7细胞磷酸酶活性的影响... 28

2.2.3 转录组测序结果概况... 29

2.2.4 差异表达基因数目分析... 30

2.2.5 qRT-PCR验证基因表达模式... 32

2.2.6 差异表达基因的功能富集分析... 35

2.2.7 E. tarda感染诱导的免疫相关差异表达基因分析... 38

2.2.8 免疫相关差异表达基因的蛋白相互作用网络分析... 41

2.2.9 E. tarda感染对RAW264.7细胞NF-κB p65磷酸化水平的影响... 42

2.3 讨论... 43

第三章 迟缓爱德华氏菌诱导的小鼠细胞因子表达模式研究... 47

3.1 实验材料与方法... 47

3.1.1 实验动物、细胞和菌株... 47

3.1.2 实验试剂与仪器... 47

3.1.3 E. tarda感染小鼠模型建立... 49

3.1.4 通过抗体芯片检测细胞因子表达情况... 49

3.1.5 差异表达细胞因子的功能富集分析... 50

3.1.6 细胞因子重组蛋白对E. tarda胞内感染的影响... 50

3.1.7 干扰细胞因子表达对E. tarda胞内感染的影响... 51

3.1.8 RNA提取和cDNA合成... 52

3.1.9 qRT-PCR.. 53

3.1.10 NO生成量检测... 53

3.2 实验结果... 54

3.2.1 E. tarda感染小鼠后的组织侵染情况... 54

3.2.2 E. tarda感染诱导的小鼠细胞因子表达情况... 55

3.2.3 差异表达细胞因子的功能富集分析... 58

3.2.4 差异表达细胞因子相互作用网络分析... 59

3.2.5 差异表达细胞因子对E. tarda胞内感染的影响... 59

3.2.6 干扰小鼠IL-6基因表达对E. tarda胞内感染的影响... 62

3.2.7 干扰牙鲆TNF-α基因表达对E. tarda胞内感染的影响... 63

3.3 讨论... 66

第四章 牙鲆趋化因子CXCL10的免疫功能研究... 69

4.1 实验材料与方法... 69

4.1.1 实验动物和菌株... 69

4.1.2 实验试剂与仪器... 69

4.1.3 序列分析和蛋白质结构预测... 71

4.1.4 qRT-PCR检测PoCXCL10表达情况... 72

4.1.5 重组蛋白表达菌株的构建... 74

4.1.6 重组蛋白rPoCXCL10、rPoCXCL10M和rSumo的表达纯化... 78

4.1.7 rPoCXCL10与细菌的结合... 80

4.1.8 rPoCXCL10对细菌生长的影响... 80

4.1.9 rPoCXCL10杀菌活性检测... 81

4.1.10 趋化活性检测... 81

4.1.11 rPoCXCL10对外周血白细胞（PBLs）基因表达的影响... 82

4.1.12 P13与细菌的结合... 83

4.1.13 P13的杀菌活性检测... 83

4.1.14 rPoCXCL10和rPoCXCL10M与PBLs的结合... 83

4.1.15 rPoCXCL10和rPoCXCL10M对PBLs内吞E. tarda的影响... 84

4.1.16 rPoCXCL10和rPoCXCL10M对牙鲆抗细菌感染的影响... 85

4.2 实验结果... 85

4.2.1 PoCXCL10的序列分析... 85

4.2.2 PoCXCL10的基因表达分析... 86

4.2.3 rPoCXCL10结合细菌和细菌细胞壁成分... 88

4.2.4 rPoCXCL10对细菌生长和存活的影响... 89

4.2.5 rPoCXCL10的趋化和免疫调节活性... 92

4.2.6 来源于PoCXCL10的C端α-螺旋区域多肽的杀菌活性... 94

4.2.7 rPoCXCL10M的趋化活性... 97

4.2.8 rPoCXCL10M的杀菌活性... 98

4.2.9 rPoCXCL10和rPoCXCL10M对牙鲆抗细菌感染免疫的影响... 100

4.3 讨论... 102

第五章 结论与展望... 105

参考文献... 109

附 录... 121

致 谢... 123

作者简历及攻读学位期间发表的学术论文与研究成果... 125