毒素-抗毒素系统SzeTA对高致病性2型猪链球菌89K毒力岛的稳定作用及其机制研究

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that caused the new emerging human infectious disease of streptococcal toxic shock syndrome (STSS) with high mortality. Our research group previously determined the whole genomic sequences of two Chinese SS2 epidemic isolates, and identified a novel pathogenicity island (PAI) designated 89K that is specific to the epidemic SS2 strains. Recently, we demonstrated that the 89K PAI can spontaneously excise from the host chromosome and its both ends join to form an extrachromosomal circular excision product. The precise excision is mediated by an 89K-borne integrase through site-specific recombination between the 15 bp perfect direct repeats flanking the island. To further investigate the function the 89K PAI, we attempted to cure this island from SS2 wild type strain 05ZYH33 to generate an 89K-negative variant but without success. Further bioimformatics analysis revealed a functional toxin-antitoxin system (TA system) designated SzeTA locates in the 89K PAI. The TA systems were recently identified as a kind of key factors that can promote the stability of mobile genetic elements such as plasmids and genomic islands. In order to evaluate the stabilization of the SzeTA system in the maintenance of the 89K PAI, and to investigate the whole function of the 89K PAI in SS2, the SzeTA system was specifically designed to be knocked out firstly in this study, then the 89K PAI was engineered to be tagged with a blue-white screening marker by inserting the bgaB gene cassette. Efforts would be then made to cure the 89K PAI from SS2, and cellular and animal experiments would be performed to assess the effect of 89K deletion on the phenotype of SS2. Finally, the SzeTA's mechanism of stabilization is expected to be illuminated by analyzing the toxic activity of the SzeT toxin and the antagonism of the SzeA antitoxin. The results of the present project will not only improve our understand of the SzeTA systems, but also lead to a better comprehension of the high pathogenicity and molecular evolution of this important pathogen. Furthermore, the 89K-negative variant may be developed as a potential vaccine candidate strain.

2型猪链球菌（SS2）是一种重要的新发人畜共患病原菌。课题组前期研究发现我国高致病性SS2上特有的89K毒力岛可以从宿主菌染色体上自发地切离并形成环形质粒样分子。为深入研究89K毒力岛的功能，课题组试图通过消除质粒的方法消除89K，始终未果。进一步分析发现89K上存在一个毒素-抗毒素（TA）系统SzeTA，而TA系统是近年证实的一类能稳定质粒和基因组岛等遗传元件的功能系统。为揭示SzeTA对89K毒力岛的稳定作用，本项目拟先通过同源重组敲除SzeTA系统，然后在89K上引入蓝白斑筛选标记，诱导筛选89K缺失株，并利用细胞和动物实验评价89K消除对SS2表型的影响，最终通过分析SzeT毒素的活性和SzeA抗毒素的拮抗作用从分子水平揭示SzeTA的稳定机制。本研究结果将极大地丰富对SzeTA系统的认识，并为SS2高致病性和遗传进化研究提供有益线索，同时89K缺失株也有望发展成为新的候选疫苗。

2型猪链球菌（Streptococcus suis serotype 2, 简称SS2）是一种新发的人兽共患病原体，不仅给全世界养猪业造成巨大经济损失，而且严重威胁公共卫生安全。近年我国突发的两次SS2大规模流行和链球菌中毒性休克综合征（Streptococcal toxic shock syndrome, STSS）这一新的临床类型的出现，提示我国流行的SS2菌株发生了毒力变异。我们前期研究发现我国高致病性SS2流行菌株含有一独特的毒力岛89K，该毒力岛可以从宿主菌染色体上自发切离并形成环形的质粒样分子，并进一步通过自身编码的IV型分泌系统作用，水平转移至不含89K毒力岛的其他菌株。为深入研究89K毒力岛的功能，我们曾试图通过消除质粒的方法消除89K，始终未能成功。本项目在前期研究工作的基础上，鉴定出89K毒力岛上存在一个有活性的毒素-抗毒素系统（toxin-antitoxin system）SezAT，该系统对89K毒力岛具有稳定作用。在细菌正常分裂时，抗毒素SezA能结合毒素SezT形成复合物，从而抑制毒素SezT对细菌的致死作用。当细菌分裂导致子代细菌中89K丢失时，不稳定的抗毒素SezA被降解，而游离出来的毒素SezT则发挥作用，消耗细菌细胞壁合成前体物质UDPAG，导致丢失89K毒力岛的子代细菌细胞壁合成受阻而死亡，凭借这种分裂后致死效应维持89K毒力岛在细菌中的稳定传代。在通过同源重组破坏SezAT系统这一稳定元件后，我们通过Cre/loxP位点特异性重组系统成功消除了89K毒力岛，并通过细胞实验和动物实验证实了89K毒力岛对于细菌的致病性至关重要。本项目研究结果不仅丰富了SezAT系统的认识，同时促进了我国高致病性SS2流行病学的分子机制认识，此外89K毒力岛缺失突变株也有望发展成为新的候选疫苗。. 另外，在本项目的支持下，我们还从铜绿假单胞菌中鉴定出一个类似于89K毒力岛的可移动遗传元件——前噬菌体pp3，可在细菌间进行水平转移；前期还从pp3中鉴定出一个有活性的毒素抗毒素系统HicAB，发表的两篇相关SCI论著均为基金第二标注。. 本项目研究成果共发表论文7篇，其中SCI论著5篇（2篇> 3.0），中文2篇；2014年获国家科技进步二等奖1项；培养硕士研究生2名。