Distribution of Plasmids in Distinct Leptospira Pathogenic Species.
Distribution of Plasmids in Distinct Leptospira Pathogenic Species.
复制标题
不同钩端螺旋体病原物种中质粒的分布。
DOI：
10.1371/journal.pntd.0004220
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发表时间：
2015-11
影响因子：
3.8
通讯作者：
Qin J
中科院分区：
文献类型：
--
作者：
Wang Y;Zhuang X;Zhong Y;Zhang C;Zhang Y;Zeng L;Zhu Y;He P;Dong K;Pal U;Guo X;Qin J
钩端螺旋体病是由致病性钩端螺旋体引起的世界性人畜共患传染病。钩端螺旋体属包括至少21个物种，根据16 S rRNA基因型分为三组-病原体，非病原体和中间体。由于钩端螺旋体生长缓慢，病原体转化能力差，因此对钩端螺旋体的研究比较困难。除两条染色体外，最近还鉴定了L.问号的研究为钩端螺旋体属的基因组复杂性提供了新的见解。这些染色体外元件的序列与染色体的大尺寸、低拷贝数和高相似性在没有仔细的基因组组装的情况下对分离和检测它们提出了挑战。在本研究中，在L.通过脉冲场凝胶电泳（S1-PFGE）分析后的全基因组组装结合S1核酸酶消化，对borgpetersenii血清型Ballum菌株56604进行了分析。此外，还对另外15株中国钩端螺旋体流行株（包括L. borgpetersenii，L. weilii和L.成功地分离和鉴定了问号，独立于基因组序列数据。Southern印迹杂交与染色体外元件特异性探针，指定为lcp 1，lcp 2和lcp 3-rep，进一步证实了它们的发生作为染色体外元件。在15个供试菌株中，13个菌株共检测到24个质粒，其中11个质粒可与lcp 1-rep探针杂交，11个质粒可与lcp 2-rep探针杂交，2个质粒可与lcp 3-rep探针杂交。它们都不可能是物种特异性的。LCP 1，LCP 2，和LCP 3-REP基因与钩端螺旋体物种基因组的非冗余蛋白质数据库的Blastp搜索表明，它们的同源序列广泛分布在病原体的分支，但不是非病原体或中间体。这些结果表明，质粒广泛分布在钩端螺旋体物种，并进一步阐明其生物学意义可能有助于我们了解致病性螺旋体的生物学和感染性。钩端螺旋体病是由钩端螺旋体属内的多种致病物种引起的，是影响发达国家和发展中国家的世界性公共卫生问题。2003年，致病性钩端螺旋体56601株全基因组测序的完成，开启了这一特异病原体的基因组研究，加快了此后的基因组测序速度。病原菌L. interrogans和L.波氏花属biflexa和中间L. licerasiae，代表钩端螺旋体属的3个系统发育群，无疑促进了对这种生物遗传复杂性的理解。最近公布了300多个菌株的基因组草图。这些序列的分析将为了解钩端螺旋体科的进化和对不同环境的适应提供宝贵的信息。染色体外复制子对于细菌之间遗传信息的交流是重要的。尽管2003年第一个钩端螺旋体全基因组测序成功，但在致病性钩端螺旋体的全基因组序列中尚未发现这些小的自身复制子。2014年前的审讯如何有效地鉴定钩端螺旋体细胞中小分子自动复制子的存在是一个技术难题，阻碍了研究人员充分了解钩端螺旋体基因组的复杂性和多样性。本研究采用S1-PFGE技术对15株中国流行的钩端螺旋体进行了小分子自动复制子的鉴定。进一步分析表明，这些质粒广泛存在于钩端螺旋体物种中。这项研究，结合最近可用的基因组测序数据，将有助于我们阐明钩端螺旋体基因组的多样性，并更深入地了解这一独特的生物进化支的进化前景。
Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups—pathogens, non-pathogens, and intermediates—based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological significance might contribute to our understanding of biology and infectivity of pathogenic spirochetes. Leptospirosis, caused by a diversity of pathogenic species within the genus Leptospira, is a worldwide public health problem affecting both developed and developing countries. In 2003, the whole genome sequencing of pathogenic Leptospira interrogans strain 56601 opened the genomic research of this specific pathogen and accelerated genomic sequencing speed afterwards. The availability of whole genome sequences of the pathogenic species L. interrogans and L. borgpetersenii, saprophyte L. biflexa and the intermediate L. licerasiae, which represented three phylogenetic groups of Leptospira spp., has undoubtedly facilitated the understanding of the genetic complexity of this organism. Genome drafts of more than 300 strains have been released recently. The analysis of these sequences will provide invaluable information for understanding the evolution and adaption to various environment of the Leptospiraceae. Extrachromosomal replicons are important for communication of genetic information between bacteria. Despite first leptospiral genome was sequenced in 2003, these small auto-replicons, however, were not identified in the whole genome sequence of pathogenic L. interrogans until 2014. How to effectively identify the presence of small auto-replicons in Leptospira cell is a technical challenge which hinders researchers in fully understanding the complexity and diversity of Leptospira genomes. In this study, we report identification of small auto-replicons in 15 Leptospira strains endemic in China by S1-PFGE analysis, independent of genome sequence assembly. Further analysis suggested that these plasmids are widely presented in Leptospira species. The study, combined with the recently available genomic sequencing data, will help us to elucidate the diversity of Leptospira genomes and a deeper insight into evolutionary perspective of this unique clade of organism.