Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
Characterization and Functional Analysis of atl, a Novel Gene Encoding Autolysin in Streptococcus suis
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DOI：
10.1128/jb.06231-11
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发表时间：
2012-03-01
影响因子：
3.2
通讯作者：
Lu, Cheng-Ping
中科院分区：
文献类型：
--
作者：
Ju, Cun-Xiang;Gu, Hong-Wei;Lu, Cheng-Ping
猪链球菌2型（S. suis 2）是引起败血症和脑膜炎的重要的猪和人类病原体。一个新的基因，命名为atl，编码一个主要的自溶素。猪2型强毒株HA 9801的分离鉴定及生物学特性研究。Atl蛋白含有1，025个氨基酸，预测分子量为113 kDa，并具有保守的N-乙酰胞壁酰-L-丙氨酸酰胺酶结构域。重组Atl在大肠杆菌中表达，其溶菌和纤连蛋白结合活性通过酶谱和蛋白质亲和印迹证实。在HA 9801的十二烷基硫酸钠提取物中显示两条溶菌带，而atl失活突变体中不存在这两条溶菌带。突变株的细胞链比亲株的细胞链更长。在自溶试验中，HA 9801的光密度（OD）值下降到初始值的20%，而突变株几乎没有自溶活性。与亲本菌株相比，atl突变体的生物膜容量降低了约30%。在斑马鱼感染模型中，突变株的50%致死剂量增加至5倍。此外，atl突变株对HEp-2细胞的粘附性比亲本菌株低50%。基于对重组Atl的功能分析和观察到的atl失活对HA 9801的影响，我们得出结论，Atl是HA 9801的主要自溶素。它参与细胞自溶、子细胞分离、生物膜形成、纤连蛋白结合活性、细胞粘附和HA 9801的发病机制。
Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-L-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced similar to 30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.