高致病性2型猪链球菌毒素-抗毒素系统SezAT的鉴定与活性研究

Identification and activity assay of the SezAT toxin-antitoxin system of highly pathogenic Streptococcus suis serotype 2

摘要:

【目的】对我国高致病性2型猪链球菌05ZYH33基因组的89K毒力岛序列进行生物信息学分析, 发现存在一对与化脓链球菌Epsilon-zeta (ε-ζ)同源的Ⅱ型毒素-抗毒素系统(Toxin-antitoxin system, TA)——SezAT, 推测该系统具有稳定89K毒力岛使其不易丢失的作用。验证SezAT为有活性的TA系统。【方法】对SezAT进行了生物信息学分析; RT-PCR验证SezAT共转录特性; 在大肠杆菌中选择性地诱导表达毒素蛋白SezT和抗毒素蛋白SezA; 最后通过同源重组技术敲除SezAT系统。【结果】sezAT由同一操纵子控制, SezT可抑制细菌生长, SezA可中和SezT的毒性作用, 同源重组成功获得sezT敲除突变株。【结论】证实SezAT为一对有活性的毒素-抗毒素(TA)系统, 为进一步研究SezAT可能发挥稳定89K毒力岛的功能, 同时获得89K毒力岛缺失突变株并深入认识89K在我国高致病性SS2中的作用奠定了基础。

Abstract:

[Objective] Bioinformatics analysis revealed that the 89K pathogenicity island (PAI) of highly pathogenic Streptococcus suis serotype 2 (SS2) in China encodes a putative type II toxin-antitoxin (TA) system named SezAT, which is homologous to the epsilon-zeta system from S. pyogenes. SezAT is presumed to be requisite for the stability of the 89K PAI in SS2. To confirm the SezAT system is a functional TA system. [Methods] The sequence characteristics of SezAT were subjected to further bioinformatics analysis. RT-PCR was performed to analyze the transcriptions of the sezAT locus and the flanking genes. The SezT toxin and SezA antitoxin proteins were selectively overexpressed in Escherichia coli. Deletion mutagenesis was carried out to obtain a SezAT-deficient mutant. [Results] Bioinformatics analysis and RT-PCR results suggest that sezAT are in the same operon. Overexpression of SezT led to severe growth inhibition of the host bacteria, while this toxicity was counteracted by the expression of SezA. Finally, the toxin-encoding gene sezT was successfully knockout by allelic replacement. [Conclusion] All of these results suggest that SezAT is an activated toxin-antitoxin (TA) system. Moreover, our results provide foundations for investigating the potential stabilized effect of SezAT in the 89K PAI, and screening an 89K-negative mutant to better understand the pathopoiesis of 89 K in highly pathogenic SS2.