三个动物源性链球菌新种的发现、基因组及毒力基因分析

发布时间：2018-08-27 12:36

【摘要】：本课题围绕“动物源病原体的发现及其对人类致病性”这一科学问题,以我国青藏高原喜马拉雅旱獭呼吸道携带的病原体谱系为研究目标,将现场流行病学调查与实验室检测分析相结合,采用PacBio高通量测序技术、微生物培养组学技术和未知新病原鉴定技术相结合,对喜马拉雅旱獭呼吸道菌群开展研究,分离获得三个链球菌新种,分别命名为旱獭链球菌(Streptcoccus marmotae sp.nov.),喜马拉雅链球菌(Streptcoccus himalayana sp.nov.)和耐盐链球菌(Streptcoccus halotolerans sp.nov.)。通过研究初步了解了喜马拉雅旱獭呼吸道菌群分布特征;明确了三个链球菌新种的准确分类地位以及与近源种的生物系统学关系;并对其全基因组特征、毒力相关因子开展了比较基因组学的分析。本课题对喜马拉雅旱獭呼吸道样本采用扩增16S rRNA序列,然后克隆转化,blast比对,对其呼吸道菌群展开分析。结果表明:链球菌属是喜马拉雅旱獭呼吸道的主要菌群,占菌群结构的49.8%;其次为埃希菌属和韦荣菌属,分别占菌群结构的11.1%和8.5%;此外,在所有未知新种的克隆中,疑似链球菌属新种的克隆数占69.1%,提示喜马拉雅旱獭呼吸道菌群中分布大量未知链球菌新种,为进一步分离培养提供了理论依据。以此为线索,我们应用微生物培养组学方法对75份喜马拉雅旱獭气管黏膜标本进行筛查,成功分离到三个疑似链球菌新种。经形态学观察、生化反应比较,并结合分子生物学鉴定,该三个未知新种均归属为链球菌属,根据三个未知新种的特征,依照国际微生物命名标准,分别将其命名为旱獭链球菌(S.marmotae sp.nov.),喜马拉雅链球菌(S.himalayana sp.nov.)和耐盐链球菌(S.halotolerans sp.nov.)。三个链球菌新种在血平板上37℃培养24 h后形成直径0.5-1.0 mm,灰白色、圆形隆起、湿润、边缘整齐的针尖状菌落,a溶血。单个菌体呈圆形或椭圆形,数个菌体排列呈链状,革兰染色阳性,无芽胞,无鞭毛。三个链球菌新种均在22、30、37、42°C培养时生长,4°C、15°C时不生长。其中喜马拉雅链球菌和旱獭链球菌在含2.5%NaCl的脑心羊血培养基上生长,在含4.5%、6.5%NaCl的脑心羊血培养基上不生长。但是耐盐链球菌在含9.0%NaCl的脑心羊血培养基上仍可以生长。三个链球菌新种的生化反应结果明显区别于同属内其他近源种。基于16S rRNA序列、3个保守基因(sodA、rpoB和groEL)和全基因组的系统发生关系分析表明,旱獭链球菌与S.suis,S.ovis和S.minor亲缘关系较近,归属为链球菌属猪链群;喜马拉雅链球菌与S.acidominimus,S.cuniculi和S.marmotae有较近的进化关系,也属于链球菌属猪链群;耐盐链球菌与变异群的S.thoraltensis,S.hyovaginalis聚在一起,归属为链球菌属变异群。通过与GenBank中其他链球菌已知种进行DNA-DNA在线杂交,发现三个链球菌新种与其他链球菌已知种相似性均低于70%。旱獭链球菌基因组全长2,322,791 bp,G+C含量为41.6 mol%,编码2,377个基因,携带58个tRNAs,12个rRNAs,2个质粒(大小分别为3,762 bp和5,997bp),11个基因组岛。喜马拉雅链球菌基因组全长2,275,471 bp,G+C含量为41.3mol%,编码2,195个基因,携带57个tRNAs,12个rRNAs,6个基因组岛。耐盐链球菌基因组全长1,823,556 bp,G+C含量为39.9mol%,编码2,068个基因,携带68个tRNAs,18个rRNAs,9个基因组岛。通过将三种链球菌新种的基因组序列与VFDB数据库收录的毒力基因进行BLAST比对分析,结果表明Has(Hyaluronic acid capsule),ClpC(Caseinolytic protease C)和ClpE(Caseinolytic protease E)是三个链球菌新种共有的毒力因子;作为肺炎链球菌主要侵袭力的表面黏附素PsaA(pneumococcal surface adhesin A)和PavA(pneumococcal adherence and virulence factor A),在旱獭链球菌和喜马拉雅链球菌中都存在;除此以外,VFDB数据库中编码细胞毒素、自溶素的基因在三个链球菌新种基因组中也存在,三个链球菌新种是否对动物和人具有致病性,其致病机制如何,是我们接下来要进行的课题研究。
[Abstract]:Focusing on the scientific problem of "discovery of animal-derived pathogens and their pathogenicity to humans", this paper takes the respiratory tract pathogen pedigree of Himalayan marmots from the Qinghai-Tibet Plateau as the research object, combines field epidemiological investigation with laboratory detection and analysis, and adopts PacBio high-throughput sequencing technology to study the microbial culture group. Three new strains of Streptococcus were isolated from the respiratory tract of Marmota himalayana sp. nov., Streptcoccus himalayana sp. Nov. and Streptcoccus halotolerans sp. Nov. respectively. The distribution characteristics of respiratory tract microflora of Himalayan marmots have been preliminarily understood, the accurate taxonomic status of three new species of Streptococcus and the relationship between the three new species and their biological systems have been clarified, and the comparative genomics analysis of the whole genome characteristics and virulence-related factors has been carried out. 16S rRNA sequence was amplified, cloned and transformed, and blast was used to analyze the respiratory tract microflora. The results showed that Streptococcus was the main microflora in the respiratory tract of Himalayan marmots, accounting for 49.8% of the microflora structure, followed by Escherichia coli and Virginia, accounting for 11.1% and 8.5% of the microflora structure, respectively. In addition, among all the clones of unknown new species, it was suspected. The clones of new species of Streptococcus accounted for 69.1%, suggesting that a large number of new species of unknown Streptococcus were distributed in the respiratory tract flora of Himalayan marmots, providing a theoretical basis for further isolation and culture. Based on this clue, 75 tracheal mucosa specimens of Himalayan marmots were screened by microbiology and three were successfully isolated. A new species of suspected Streptococcus was identified as S. marmotae sp. Nov. and S. himalayana sp. according to the characteristics of the three unknown new species and according to the international microbiological nomenclature standards, they were named S. marmotae sp. Nov. and S. himalayana sp. respectively. S. halotolerans sp. Nov. and S. halotolerans sp. Nov. Three new species of Streptococcus were cultured on the blood plate at 37 C for 24 hours and then formed 0.5-1.0 mm in diameter, gray-white, round protuberance, moist, well-margined, needle-shaped colony, a hemolysis. A single bacteria was round or oval, several bacteria arranged in a chain shape, Gram-positive, sporeless, flagella-free. Streptococcus himalayanus and Streptococcus Marmota grew on the medium containing 2.5% NaCl and did not grow on the medium containing 4.5% and 6.5% NaCl. But Streptococcus halotolerans could still grow on the medium containing 9.0% NaCl. The results of biochemical reactions of three new Streptococcus species were significantly different from other near source species. Based on 16S rRNA sequences, 3 conserved genes (sodA, rpoB and groEL) and genome wide phylogenetic relationships showed that Streptococcus marmos was closely related to S.suis, S.ovis and S.minor and belonged to Streptococcus group *; Himalaya. Streptococcus has a closer evolutionary relationship with S.acidominimus, S.cuniculi and S.marmotae, and also belongs to Streptococcus * group. Streptococcus salt resistant Streptococcus is clustered together with S.thoraltensis and S.hyovaginalis of the mutant group and belongs to the group of Streptococcus mutans. By means of DNA-DNA online hybridization with other known species of Streptococcus in GenBank, three Streptococcus were found. The genome of Streptococcus Marmota was 2,322,791 BP in length, with a G+C content of 41.6 mol%, encoding 2,377 genes, carrying 58 tRNAs, 12 rRNAs, 2 plasmids (3,762 BP and 5,997 BP respectively) and 11 genomic islands. The genome of Streptococcus himalayae was 2,275,471 BP in length and 41.3 m in G+C content. Ol%, encoding 2,195 genes, carrying 57 tRNAs, 12 rRNAs, 6 genomic islands. The genome of S. halotolerans was 1,823,556 BP in length, containing 39.9 mol% of G + C, encoding 2,068 genes, carrying 68 tRNAs, 18 rRNAs, and 9 genomic islands. BLAST ratios were made between the genomic sequences of the three new species of S. halotolerans and the virulence genes in the VFDB database. The results showed that Has (Hyaluronic acid capsule), ClpC (Caseinolytic protease C) and CPE (Caseinolytic protease E) were the common virulence factors of three new species of Streptococcus, and the surface adhesins PsaA (pneumococcal surface adhesin A) and PAVA (pneumococcal adherence and virulence factor) were the main invasive factors of Streptococcus pneumoniae. In addition, cytotoxins are encoded in the VFDB database, and autolysin genes are also present in the genomes of three new species of Streptococcus. Whether the three new species of Streptococcus are pathogenic to animals and humans, and how their pathogenic mechanisms are, are the topics we will study next.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.611

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