乙酰基转移酶毒素SsGNAT影响猪链球菌的黏附能力

Acetyltransferase toxin SsGNAT affects adhesion of Streptococcus suis

【目的】猪链球菌(Streptococcus suis)是一种重要的人畜共患病病原,本研究旨在鉴定猪链球菌乙酰基转移酶毒素类毒素-抗毒素系统,并对其毒素功能进行分析,探讨猪链球菌毒素-抗毒素系统在细菌感染过程中发挥的作用。【方法】前期预测猪链球菌血清型5型菌株HN105基因组中假定的Ⅱ型毒素-抗毒素系统。进一步鉴定毒素-抗毒素系统的活性并分析该毒素-抗毒素系统的遗传进化关系;运用Western blotting揭示毒素在猪链球菌内的表达情况。同时以HN105为亲本株,构建毒素缺失株、抗毒素缺失株以及毒素-抗毒素缺失株,研究该系统对猪链球菌黏附能力、生物被膜形成能力、抗吞噬与胞内存活能力的影响。【结果】预测并鉴定出猪链球菌血清型5型菌株HN105基因组中DF184_RS00980-DF184_RS00985编码的乙酰基转移酶(Gcn5-related N-acetyltransferase,GNAT)毒素类毒素-抗毒素系统,根据保守结构域将该系统命名为SsMarR-SsGNAT。SsGNAT毒素在大肠杆菌(Escherichia coli)的周质间隙发挥毒性作用,且抗毒素可以中和毒素的毒性;猪链球菌SsGNAT毒素与目前已鉴定的乙酰基转移酶毒素氨基酸序列一致性较低且亲缘关系较远;SsGNAT毒素在猪链球菌内存在被切割的现象,且抗毒素表达量降低可上调SsGNAT毒素的表达量;SsGNAT毒素对猪链球菌的生物被膜形成能力、抗吞噬与胞内存活能力无影响,但可影响猪链球菌对人喉癌上皮细胞(human laryngeal cancer epithelial cells,Hep-2)和人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMEC)的黏附能力。【结论】发现并鉴定了猪链球菌中新的Ⅱ型毒素-抗毒素系统SsMarR-SsGNAT,SsGNAT毒素在大肠杆菌周质间隙发挥毒性作用,缺失后导致细菌对细胞的黏附水平显著上升,该发现为进一步揭示猪链球菌的致病机制提供参考。

[Objective]Streptococcus suis is a major zoonotic pathogen.This study aims to characterize the acetyltransferase toxin of the toxin-antitoxin system in Streptococcus suis and analyze the functions of the toxin,which may contribute to exploring the role of the toxin-antitoxin system in the infection of S.suis.[Methods]We first predicted the putative typeⅡtoxin-antitoxin system in the genome of S.suis serotype 5 strain HN105.We further determined the activity and analyzed the phylogenetic relationship of the toxin-antitoxin system.Western blotting was employed to determine the expression of the toxin in S.suis.We constructed the toxin-deficient strain,antitoxin-deficient strain,and toxin-antitoxin-deficient strain with HN105 as the parental strain to explore the effects of the system on the adhesion,biofilm formation,anti-phagocytosis,and intracellular survival of S.suis.[Results]The Gcn5-related N-acetyltransferase(GNAT)toxin of the toxin-antitoxin system encoded by DF184_RS00980-DF184_RS00985 in the genome of the strain HN105 was predicted and identified,and the system was named SsMarR-SsGNAT based on the conserved domain.SsGNAT toxin exerted toxicity in the periplasmic space of Escherichia coli,and the antitoxin could neutralize the toxicity.SsGNAT toxin showed low amino acid sequence identity and distant genetic relationship with other acetyltransferase toxins.SsGNAT toxin was cleaved in S.suis,and the reduced expression of antitoxin upregulated the expression of SsGNAT toxin.SsGNAT toxin had no effect on the biofilm formation,anti-phagocytosis or intracellular survival of S.suis,while it affected the adhesion of S.suis to human laryngeal cancer epithelial cells(Hep-2)and human brain microvascular endothelial cells(HBMEC)cells.[Conclusion]We discovered and identified the novel typeⅡtoxin-antitoxin system SsMarR-SsGNAT in S.suis.SsGNAT toxin exerted toxicity in the periplasmic space of E.coli,and its deletion promoted bacterial adhesion to cells.The findings provide a reference for further unraveling the pathogenic mechanism of S.suis.