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Investigation of a Novel DNase of Streptococcus suis Serotype 2

机译：猪链球菌血清型2的新型DNase的研究

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A secreted nuclease, SsnA, was identified in the virulent Streptococcus suis isolate SX332 and subsequently in each of the type strains of capsular serotypes 1 through 9. Screening of 258 porcine clinical isolates from surface (nasal mucosa or palatine tonsil) or internal (joint, brain or other internal organ) locations revealed a significant relationship (P < 0.001) between expression of nuclease and isolation from an internal site. A 3,126-bp gene, ssnA, was identified from a phenotypically nuclease-negative pGh9:ISS1 insertion mutant, and analysis of the predicted SsnA sequence revealed a 35-amino-acid (aa) secretion signal sequence, a 22-aa DNA-binding domain, and a typical gram-positive cell wall sorting motif. A requirement of Ca2+ and Mg2+ for SsnA activity was determined, and the substrate specificity was found to be for single- and double-stranded linear DNA. Reverse transcription-PCR experiments revealed that ssnA is expressed throughout all stages of S. suis growth, and Western blots with porcine anti-S. suis immune sera against a recombinant, truncated SsnA derivative (rSsnAΔ) confirmed that SsnA is expressed in vivo. Furthermore, anti-rSsnAΔ antibodies were sufficient to neutralize SsnA activity. Analyses of subcellular fractions of SX332 and derived mutants, on DNA-containing polyacrylamide gels and by Western blotting, suggest that SsnA is cell wall located.
机译：在强力猪链球菌分离株SX332中鉴定出一种分泌的核酸酶SsnA，随后在荚膜血清型1至9的每种类型菌株中鉴定出了这种酶。从表面（鼻粘膜或p鱼）中筛选了258株猪临床分离株扁桃体）或内部（关节，大脑或其他内部器官）位置显示了核酸酶的表达与从内部位点分离之间的显着关系（ P <0.001）。从表型上的核酸酶阴性pGh9：IS S1 插入突变体中鉴定出一个3,126 bp的基因 ssnA ，对预测的SsnA序列进行分析后发现了一个35个氨基酸的酸（aa）分泌信号序列，22-aa DNA结合结构域和典型的革兰氏阳性细胞壁分选基序。确定了Ca 2 + 和Mg 2 + 对SsnA活性的要求，并且发现底物特异性针对单链和双链线性DNA。逆转录PCR实验表明， ssnA 在 S的所有阶段均表达。猪生长，并用猪抗 S蛋白进行Western印迹分析。重组截短的SsnA衍生物（rSsnA Δ）的suis 免疫血清证实SsnA在体内表达。此外，抗rSsnA Δ抗体足以中和SsnA活性。在含DNA的聚丙烯酰胺凝胶上并通过Western印迹分析SX332的亚细胞部分和衍生的突变体，表明SsnA位于细胞壁上。

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