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两株深海来源蜡样芽孢杆菌群细菌的感染与毒力特征研究
赵炎
Subtype博士
Thesis Advisor孙黎
2020-05
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 维德曼芽孢杆菌 蜡样芽孢杆菌 毒力 细胞焦亡
Abstract

蜡样芽孢杆菌群(Bacillus cereus group)细菌分布十分广泛,主要分布在土壤、腐烂有机质、水体等环境,最近发现其在深海等海洋环境中也有分布。蜡样芽孢杆菌群细菌可形成内生芽孢而具有对环境压力顽强的抵抗力。蜡样芽孢杆菌群细菌是重要的食源性病原,引起肠道疾病如腹泻综合症和非肠道疾病如脓毒症等。本研究中,我们从深海热液和冷泉采集的样品中,分离鉴定出两株蜡样芽孢杆菌群细菌,分别命名为维德曼芽孢杆菌(Bacillus wiedmanniiSR52和蜡样芽孢杆菌(Bacillus cereusH2。通过基因组学分析、体内体外实验等方法,研究了这两株深海来源细菌的感染与毒力特征。

菌株SR52分离自冲绳海槽一个热液区的阿尔文虾(Alvinocaris longirostris)的鳃。系统发育分析发现细菌SR52与维德曼芽孢杆菌FSL W8-0169亲缘关系最近,而维德曼芽孢杆菌是蜡样芽孢杆菌群中的一员。SR52好氧,具有运动性,能够形成内生芽孢,可以在高达9%的NaCl浓度下生长。SR52有一个环状染色体,大小为5448361 bp,编码5709个基因。SR52还有一个环状质粒,大小为137592 bp,编码189个基因。SR52染色体预测有297个毒力基因,包括编码肠毒素和溶血素的基因。染色体中有14rRNA操纵子、107tRNA5sRNA,质粒仅有7tRNASR52的染色体中发现有13个基因岛。与维德曼芽孢杆菌FSL W8-0169相比,SR52的基因组表现出一些显著特点,尤其是非编码RNA和基因岛数量增加明显。体内研究表明,通过腹腔注射小鼠(Mus musculus)时,SR52在小鼠组织中扩散并导致小鼠的急性死亡。体外研究表明,SR52有溶血活性。

另一株细菌H2是从南海冷泉的潜铠虾(Shinkaia crosnieri)的刚毛中分离得到的。通过体内感染实验,我们发现肌肉注射大菱鲆(Scophthalmus maximus)或腹腔注射小鼠时,细菌H2均在宿主组织中感染,并引起急性死亡。体外感染实验表明,细菌H2引起上皮细胞和巨噬细胞死亡。H2感染的巨噬细胞以依赖NLRP3的方式发生细胞焦亡。H2感染激活了巨噬细胞的p38JNKERK通路,但只有JNK参与了H2诱导的细胞焦亡。H2感染引起胞内活性氧的增加,并且活性氧对于H2诱导的JNK激活和NLRP3炎症小体的激活都至关重要。另外,在H2感染过程中,细胞内Ca2+对于H2诱导的细胞焦亡也是不可或缺的。

本研究首次揭示了来自深海环境的两株蜡样芽孢杆菌群细菌的细胞毒性和感染能力,为蜡样芽孢杆菌的致病机制提供了新的见解。

Other Abstract

Bacillus cereus group strains are widespread in nature, mainly in soil, decaying organic matter and water. They have been found to be distributed in marine environments such as the deep sea recently. Bacillus cereus group strains are members of the most important foodborne pathogens; they have strong resistance to environmental stress becasus of their endospores and can cause gastrointestinal diseases such as diarrheal syndrome and non-gastrointestinal diseases such as sepsis. In this study, we identified two strains of the Bacillus cereus group isolated from the deep-sea hydrothermal vent and the deep-sea cold seeps, and named them Bacillus wiedmannii SR52 and Bacillus cereus H2, respectively. In addition, we expounded the characteristics of infection and virulence of these bacteria using genomics analysis and in vitro/vivo experimental studies.

Strain SR52 was isolated from the gills of shrimp Alvinocaris longirostris that dwelled on the hydrothermal vent of Okinawa Trough. SR52 is most closely related to Bacillus wiedmannii FSL W8-0169, a member of the Bacillus cereus group. SR52 is aerobic, motile, and able to form endospores. SR52 can grow in high concentrations (up to 9%) of NaCl. SR52 has a circular chromosome of 5,448,361 bp and a plasmid of 137,592 bp, encoding 5,709 and 189 genes, respectively. The chromosome contains 297 putative virulence genes, including those encoding enterotoxins and hemolysins. Fourteen rRNA operons, 107 tRNAs, and 5 sRNAs are present in the chromosome, and 7 tRNAs are present in the plasmid. SR52 possesses 13 genomic islands (GIs), all on the chromosome. Comparing to FSL W8-0169, SR52 exhibits several streaking features in its genome, notably an exceedingly large number of non-coding RNAs and GIs. In vivo studies showed that following intraperitoneal injection into mice Mus musculus, SR52 was able to disseminate in tissues and cause acute mortality. In vitro studies showed that SR52 possessed hemolytic activity.

We isolated the other bacteria, strain H2, from the setae of the galatheid crab Shinkaia crosnieri collected at a deep-sea cold seep in South China Sea. In vivo study showed that H2 was capable of tissue dissemination and causing acute mortality in mice and turbot Scophthalmus maximus following intraperitoneal/intramuscular injection. In vitro study showed that H2 caused death of epithelial cells and macrophages. Study using macrophages revealed that H2-infected cells exhibited pyroptotic death in a manner that was dependent on NLRP3. H2 infection activated p38, JNK, and ERK, but only JNK proved to participate in H2-triggered pyroptosis. The production of reactive oxygen species (ROS) increased after infection and was essential to H2-induced activation of JNK and NLRP3 inflammasome. Similarly, intracellular Ca2+ was also important to the activation of JNK and NLRP3 inflammasome during H2 infection.

This study demonstrated for the first time the cytotoxicity and infectivity of two deep-sea bacteria belonging to the Bacillus cereus group and provided new insights into the mechanism of Bacillus cereus infection.

Subject Area海洋生物学
MOST Discipline Catalogue理学 ; 理学::海洋科学
Pages109
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164654
Collection实验海洋生物学重点实验室
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