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深海热液一株枯草芽孢杆菌和一种抗脂多糖因子的鉴定与分析
谷翰杰
Subtype博士
Thesis Advisor孙黎
2019-05-17
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword深海热液 芽孢杆菌 细胞毒力 阿尔文虾 抗脂多糖因子
Abstract

枯草芽孢杆菌是研究最为透彻的革兰氏阳性细菌,其主要应用于工业开发和作为细胞分化研究的模型。迄今为止,人们对枯草芽孢杆菌的毒性知之甚少。在本研究中,我们从冲绳海槽的Iheya North热液区的水体样品中分离、鉴定出一株对鱼类和小鼠具有致死能力的枯草芽孢杆菌(G7)。G7NaCl依赖型、可运动和形成内生孢子的需氧菌。G7的基因组由包含4216133个碱基对的一个环状染色体组成,平均GC含量为43.72%G7含有4416个编码基因,COG功能分析表明,其中72.5%的基因可以被注释到已知的或预测的功能,但27.5%的编码基因不能被注释到功能。在G7的基因组中发现了1023S5S16S核糖体RNA操纵子,86tRNA14sRNA基因,50个串联重复序列,41个小卫星,1个微卫星和42个转座子序列。比较基因组分析表明,与枯草芽孢杆菌的野生型标准菌株NCIB 3610T的基因组相比,G7基因组中的许多同源基因发生了易位和倒位的现象,还存在多个插入序列,且G7基因岛(Genomic IslandsGIs)的数量是NCIB 3610T基因岛数量的两倍,在G7基因岛所覆盖的所有基因中,42.5%的基因编码的是一些功能未知的蛋白质。此外,G7的基因组中还预测到一些潜在的毒力因子,这些毒力因子可能与细菌的粘附、侵染、传播、抗吞噬和细胞内存活等功能相关。毒力实验研究表明,在人工感染后,G7能够引起鱼和小鼠的死亡,并且能抵抗鱼和小鼠血清的杀伤。进一步研究发现,G7在抵御不同动物血清杀伤的过程中表现出不同的策略:G7能有效地激活鱼血清的补体系统,但其表面坚实的细胞壁结构能抵御补体膜攻击复合物的杀伤;G7不能有效地激活小鼠血清的补体系统,进而避免了补体系统的杀伤。除此之外,G7还具有在小鼠的巨噬细胞和鱼的外周血淋巴细胞中复制的能力。综上所述,我们的研究表明G7是一株具有独特遗传特征的、有致死能力的枯草芽孢杆菌,这为深入了解深海热液系统中芽孢杆菌属的致病性提供了基础。

阿尔文虾是深海热液区的代表性物种,是一种典型的深海无脊椎动物。然而,阿尔文虾是否具有与其他无脊椎动物类似的免疫防御机制还不清楚。我们获得了来自马努斯盆地Desmos深海热液喷口附近的阿尔文虾Rimicaris sp.样品,通过转录组学分析,我们发现Rimicaris sp.表达高丰度的抗脂多糖因子(Anti-lipopolysaccharide factorALF)基因。抗脂多糖因子属于抗菌肽(Antimicrobial peptidesAMPs)家族,在浅海和陆地的无脊椎动物抵抗细菌感染的免疫防御过程中起着至关重要的作用。尽管已经从浅海和淡水甲壳动物中鉴定出大量的ALF,但深海动物ALF的免疫功能研究仍属空白。在本研究中,我们研究了阿尔文虾ALFRspALF1)的抗感染免疫功能。序列分析表明,RspALF1与已知其他物种的虾类的ALF具有51.5-62.4%的相似性,并含有保守的LPS结合结构域(LPS binding domainLBD)。重组表达的RspALF1rRspALF1)和人工合成的LBD活性小肽(ALF1P1)均能识别革兰氏阴性和革兰氏阳性细菌的细胞壁成分,包括脂多糖和葡聚糖等,并能够通过破坏细胞壁杀死多种细菌,尤其是G7等来自深海热液区的细菌。ALF1P14-37°C条件下具有较高杀菌活性,这与阿尔文虾栖息地的环境温度范围相一致。除了具有细菌杀伤活性外,ALF1P1还表现出抗真菌的活性。ALF1P1对细菌的最低抑菌浓度(Minimal inhibit concentrationMIC)为2-4 μM,对真菌的MIC4-8 μM。鱼和虾的体内实验表明,rRspALF1ALF1P1均能够有效地提高虾类抗细菌感染的能力,也均能有效地阻止细菌和病毒在鱼组织中的侵染和扩散。本研究首次对深海无脊椎动物ALF的生物学特性进行了深入研究,揭示了深海ALF的免疫学功能和抗细菌、抗病毒能力,研究结果为深海来源的高效抗菌剂发掘提供了理论基础。

Other Abstract

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we reported a B. subtilis (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough which could cause mortality in fish and mouse. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of GIs, with 42.5% of GI genes being predicted to encode hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Consistently, G7 was able to cause mortality-leading infection in fish and mice following injection, replicate in mouse macrophages and fish peripheral blood leukocytes, and resist the killing effect of serum complement, in addition to this, we found that G7 adopted different serum resistance strategies towards different animals: G7 can activate the complement system of fish successfully, but its solid cell wall structure can help it resist the killing of complement MAC; G7 can not effectively activate the mouse complement system, and thus avoid the killing. Taken together, our study indicates that G7 is a virulent B. subtilis isolate with unique genetic features, and the results provide the first molecular insight into the virulence of Bacillus species in deep-sea hydrothermal vent.

ALF (Anti-lipopolysaccharide factor) is a type of AMPs with a vital role in antimicrobial defense. Although a large amount of ALFs have been identified from neritic and fresh water crustacean species, no functional investigation of ALFs from deep-sea animals have been documented. In the present study, we characterized the immune function of an ALF molecule (named RspALF1) from the shrimp Rimicaris sp. residing in the deep-sea hydrothermal vent in Desmos, Manus Basin. RspALF1 shares 51.5%-62.4% overall sequence identities with known shrimp ALFs and contains the conserved LPS binding domain (LBD). Both recombinant RspALF1 (rRspALF1) and the LBD-derived peptide (ALF1P1) bound to the cell wall components of Gram-negative and Gram-positive bacteria and killed a wide range of bacteria, especially those from deep-sea hydrothermal field, by damaging bacterial cellular structures. The bactericidal activity of ALF1P1 was optimal and stably maintained from 4°C to 37°C, which is comparable to the ambient temperature range of the habitat of Rimicaris sp. In addition to bacteria, ALF1P1 also exhibited anti-fungal activity. ALF1P1 exhibited high killing efficiencies, which, in terms of MIC values, were ranged between 2 μM to 4 μM for bacteria and 4 μM to 8 μM for fungi. When introduced in vivo, both rRspALF1 and ALF1P1 effectively inhibited bacterial infection in shrimp and reduced the dissemination of bacterial and viral pathogens in fish. Together, these results provide the first insight into the biological property of deep-sea ALF and indicate that RspALF1 very likely plays a significant role in immune defense by functioning as a highly effective antimicrobial with a broad target range.

MOST Discipline Catalogue理学 ; 理学::海洋科学 ; 理学::生物学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156787
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
谷翰杰. 深海热液一株枯草芽孢杆菌和一种抗脂多糖因子的鉴定与分析[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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