实验海洋生物学重点实验室知识库

随着人类深海探索活动的不断增加，深海微生物，包括对哺乳动物可能具有致病能力的微生物，将不可避免地被携带至近海和陆地环境中，这对人类健康和环境安全等可能造成危害。因此，开展深海来源微生物对脊椎动物致病性的研究，并探索相关微生物疾病的免疫防控，对保障人类健康和环境安全具有重要意义。基于此，本论文的目的是研究深海来源微生物对近海和陆地脊椎动物的致病性，同时筛选、发现针对深海来源的致病微生物具有杀伤作用的抗菌因子，为深海病原微生物潜在疾病的免疫防控提供基础。

        蜡样芽孢杆菌是一种重要的条件致病菌，广泛分布于自然环境中，能够引起胃肠道和非胃肠道类型的感染。在本论文研究中，我们从马里亚纳海沟挑战者深渊区的水样中分离出一株蜡样芽孢杆菌，并命名为MB1。MB1好氧、具有运动能力并且能够形成内生芽孢，可以在无NaCl或者高达6%的NaCl浓度下生长。MB1拥有一个环状染色体和两个环状质粒（pMB1a和pMB1b），总计编码5966个基因。MB1基因组包含14组由23S、5S和16S构成的核糖体RNA操纵子、106个tRNA基因、4个sRNA基因、12个基因组岛和13个前噬菌体基因。MB1编码302个与毒力相关的基因，其中292个基因位于染色体中，10个位于pMB1b质粒。体内实验研究表明，MB1能够在大菱鲆和小鼠中引起急性致命感染，并且能够肝脏、脾脏和肾脏中扩散。体外研究表明，MB1对哺乳动物细胞具有高毒性和溶血性，并以剂量依赖性方式诱导细胞焦亡。进一步研究表明，MB1诱导细胞焦亡的过程中，caspase-1和gasdermin D被剪切激活，IL-1β和IL-18被释放至胞外。初始状态为芽孢的MB1在24h内即具有运动和溶血能力，但对大菱鲆的致病性严重减弱。然而，当芽孢在合适的条件下萌发为营养细胞，其细胞毒性得到完全的恢复。

       Cereolysin O是一种归属于胆固醇依赖性溶细胞素（CDC）家族的细胞素，也被称为溶血素I。在本研究中，我们从蜡样芽孢杆菌MB1编码的毒力基因数据库中发现了一种Cereolysin O，命名为CLO，并研究了其毒力。CLO与陆地来源的Cereolysin O序列相似性为 98.83%，与CDC家族其他溶细胞素（包括anthrolysin O、perfringolysin O和streptolysin O等）的序列相似性为54%左右。细胞实验证明，重组CLO蛋白（rCLO）具有显著的溶血活性和细胞裂解能力。基因突变研究发现， 477和479位的色氨酸对rCLO的活性至关重要，其原因可能是该部位的氨基酸残基参与rCLO与细胞膜的结合。

        针对MB1的感染能力和细胞毒性，我们尝试寻找一种能够有效抑制或直接杀灭MB1的抗菌因子，如抗菌肽。抗菌肽被认为是一类广泛存在于生物体中的天然抗生素，在先天免疫中起重要作用。在此部分研究中，我们从栖息在马努斯盆地深海热液的虾Rimicaris sp. 中发现了两种甲壳素（crustin）类抗菌肽，命名为Crus1和Crus2。 Crus1和Crus2均归属于I型Crustin，拥有一个位于羧基端的乳清酸性蛋白（WAP）结构域，在此结构域中包含由8个高度保守的半胱氨酸残基（cysteine）形成的“四二硫键核心”结构。重组Crus1（rCrus1）和Crus2（rCrus2）均对包括MB1在内的革兰氏阳性细菌具有显著的抗菌作用，并且rCrus2对部分革兰氏阴性细菌同样有抑制/杀伤作用。rCrus1能够与细菌肽聚糖和脂磷壁酸结合，并以依赖于pH和二硫键的方式有效杀死革兰氏阳性细菌。rCrus1能够造成细菌细胞膜渗漏和结构损伤，但对细菌原生质体没有影响。WAP结构域中的8个半胱氨酸残基中的任何一个被丝氨酸取代都不影响rCrus1与细菌的结合能力，但完全消除了rCrus1的杀菌活性。

        综上所述，本研究揭示了马里亚纳海沟超深渊区8,003 m处蜡样芽孢杆菌MB1的基因组结构及对陆地和近海脊椎动物的感染能力和细胞毒性，阐明了一种MB1毒力因子（CLO）的作用机制，发现了能有效对抗MB1的抗菌分子（crustin）并探索了其抗菌机制。这些结果加深了对深海致病微生物的了解，为深海微生物疾病防控提供了理论指导。

With the increase of deep-sea exploration activities, deep-sea microorganisms, including pathogenic microorganisms to mammals, will inevitably be brought into the terrestrial environments, which may cause potential harms to human health and environment safety. Therefore, it is important to study the infectivity of dee-sea microbes to vertebrates and explore the means to prevent and control the relevant microbial disease. Hence, the aim of this thesis is to examine the pathogenicity of dee-sea bacteria to mammal and teleost, thus promoting the understanding of the potential risk of dee-sea pathogenic microbes to humans and the environment. At the same time, this thesis also aims to explore antibacterial factors with bacteriacidal effect on the dee-sea pathogens, thus providing a foundation for the prevention and control of potential diseases associated with deep-sea pathogenic microorganisms.

Bacillus cereus is an important opportunistic pathogen widely distributed in the environment, which can cause gastrointestinal and in nongastrointestinal infections. In this study, we reported the isolation and characterization of a B. cereus isolate, MB1, from the Challenger Deep of the Mariana Trench. MB1 is aerobic, motile, and able to form endospores. MB1 possesses 5966 genes distributed on a circular chromosome and two plasmids. The MB1 genome contains 14 sets of 23S, 5S, and 16S ribosomal RNA operons, 106 tRNA genes, 4 sRNA genes, 12 genomic islands, 13 prophages, and 302 putative virulence genes, including enterotoxins and cytolysins. Live animal studies showed that MB1 was able to cause acute and lethal infection in fish and mice. In vitro studies showed that MB1 was highly toxic and hemolytic to mammalian cells and induced pyroptosis in a dose-dependent manner. Further studies showed that during MB1-induced pyroptosis, caspase-1 and gasdermin D were activated by cleavage, and IL-1β and IL-18 were released extracellularly. MB1 spores exhibited swimming and hemolytic capacity, but were severely attenuated in pathogenicity, which, however, was regained to the full extent when the spores germinated under suitable conditions. Taken together, these results provide new insights into the biological and pathogenic mechanism of deep-sea B. cereus.

Cereolysin O is a cytotoxin belonging to the cholesterol-dependent cytolysin (CDC) family, also known as hemolysin I. In this study, we identified a Cereolysin O (CLO) gene from the genomic database of Bacillus cereus MB1 and analyzed its cytotoxicity. CLO is 98.83% identical to the Cereolysin O of other B. cereus and ~54% identical to other cytolysins of the CDC family (including anthrolysin O, perfringolysin O and streptolysin O, etc.). Recombinant CLO (rCLO) exhibited significant hemolytic activity and cytotoxicity to mammalian cells. Gene mutation analysis showed that W477 and W479 are essential for the activity of rCLO, which may be due to the involvement of these residues in cell membrane binding.

We next tried to find an antibacterial factor, such as antimicrobial peptides (AMP), that can effectively kill MB1 or inihibit its growth. AMP are widely distributed natural antibiotics that play an important role in innate immunity. We identified two Crustin-like antimicrobial peptides, named Crus1 and Crus2, from the shrimp Rimicaris sp. inhabiting the deep-sea hydrothermal vent in Manus Basin. Both Crus1 and Crus2 belong to type I Crustin, possessing a whey acidic protein (WAP) domain in the carboxy-terminal, in which a "four-disulfide core" structure is formed by eight highly conserved cysteine (Cys) residues. Both recombinant Crus1 (rCrus1) and Crus2 (rCrus2) had significant antibacterial effects against Gram-positive bacteria including MB1, while rCrus2 was equally effective against some Gram-negative bacteria. rCrus1 bound to peptidoglycan and lipoteichoic acid, and effectively killed Gram-positive bacteria in a manner that was dependent on pH, temperature, and disulfide linkage. rCrus1 caused bacterial membrane leakage and structural damage, but had no effect on bacterial protoplasts. Serine (Ser) substitution of each of the 8 Cys residues in the WAP domain did not affect the bacterial binding capacity but completely abolished the bactericidal activity of rCrus1.

In conclusion, this study revealed the genomic structure, infectivity and cytotoxicity of Bacillus cereus at 8,003 m in the Mariana Trench, and demonstrated the cytotoxicity of a MB1 virulence factor. In addition, we identified two deep-sea AMPs with bactericidal activity towards MB1, and explored their antibacterial mechanism. These results deepen the understanding of the infection mechanism of deep-sea pathogenic microorganisms and provide theoretical guidance for the prevention and control of deep-sea microbial diseases.

目  录

第1章  绪论... 1

1.1  深海生态系统    1

1.1.1  深海环境概述... 1

1.1.2  海沟生态系统... 2

1.1.3  深海热液生态系统... 3

1.2  蜡样芽孢杆菌    5

1.2.1  蜡样芽孢杆菌生物学特征... 5

1.2.2  蜡样芽孢杆菌流行病学调查... 6

1.2.3  蜡样芽孢杆菌毒素... 7

1.2.4  深海来源的芽孢杆菌... 11

1.3  抗菌肽    12

1.3.1  抗菌肽概述... 12

1.3.2  抗菌肽作用机制... 14

1.3.3  甲壳素... 18

1.4  本文的研究目的和意义    21

第2章  蜡样芽孢杆菌MB1的分离鉴定及毒性分析... 23

2.1  实验材料和方法    23

2.1.1  菌株、细胞及实验动物... 23

2.1.2  试剂、耗材及仪器... 23

2.1.3  细菌和细胞培养... 25

2.1.4  致病菌筛选... 25

2.1.5  基本生物学特征... 26

2.1.6  基因组测序及系统发育分析... 29

2.1.7  体内感染试验... 31

2.1.8  体外感染试验... 32

2.1.9  免疫印迹... 33

2.1.10  ELISA检测... 34

2.1.11  Annexin V-FITC/ PI双染... 35

2.2  实验结果    35

2.2.1  MB1形态结构及生长特征... 35

2.2.2  MB1系统发育分析及鉴定... 38

2.2.3  MB1基因组特征... 39

2.2.4  MB1和ATCC 14579的比较基因组分析... 43

2.2.5  MB1的毒力相关基因... 45

2.2.6  MB1对大菱鲆和小鼠的致病性... 45

2.2.7  MB1的细胞毒性... 48

2.2.8  MB1诱导细胞焦亡... 49

2.2.9  MB1芽孢萌发后的活性与细胞毒性... 52

2.3  讨论    54

第3章  MB1编码的Cerolysin毒力分析... 59

3.1  实验材料和方法    59

3.1.1  试剂、耗材及仪器... 59

3.1.2  序列及结构特征分析... 60

3.1.3  基因克隆、重组质粒构建及转化... 60

3.1.4  重组蛋白原核表达及纯化... 65

3.1.5  溶血活性检测... 66

3.1.6  细胞毒性检测... 67

3.2  实验结果    67

3.2.1  CLO的序列及结构特征... 67

3.2.2  rCLO的溶血活性... 69

3.2.3  rCLO的细胞毒性... 70

3.2.4  CLO四个结构域内保守氨基酸残基对溶血活性的影响... 72

3.2.5  CLO四个结构域内保守氨基酸残基对细胞毒性的影响... 74

3.3  讨论    75

第4章  深海来源抗菌肽Crus1/Crus2对MB1以及其它细菌的杀伤作用与机制... 77

4.1  实验材料与方法    77

4.1.1  实验菌株... 77

4.1.2  试剂、耗材及仪器... 77

4.1.3  序列及结构特征分析... 78

4.1.4  基因合成/克隆，重组质粒构建及转化... 78

4.1.5  重组蛋白原核表达及纯化... 80

4.1.6  抑菌及杀菌实验... 80

4.1.7  结合实验... 82

4.1.8  细菌膜破坏检测... 82

4.1.9  原生质体相关... 84

4.1.10  圆二色谱检测... 84

4.2  实验结果    85

4.2.1  Crus1和Crus2的序列和结构表征... 85

4.2.2  Crus1和Crus2蛋白表达纯化结果... 89

4.2.3  rCrus1和rCrus2的抑菌及杀菌活性... 89

4.2.4  温度、pH和二硫键对rCrus1杀菌活性的影响... 91

4.2.5  rCrus1对细菌及细胞壁组分的结合活性... 94

4.2.6  rCrus1对细菌形态和膜完整性的影响... 96

4.2.7  rCrus1对原生质体的影响... 99

4.2.8  WAP结构域中二硫键对rCrus1杀菌活性的影响... 101

4.3  讨论    104

第5章  结论与展望... 107

参考文献... 109

附  录... 125

致 谢... 139

作者简历及攻读学位期间发表的学术论文与研究成果... 141