Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy.
PLoS One. 2013;8(2):e57775. doi: 10.1371/journal.pone.0057775. Epub 2013 Feb 28.
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.
在这项研究中，我们对黏质沙雷氏菌 MAG_5040 的酶活性进行了研究，它是一种与金黄色葡萄球菌 SNase 具有同源性的依赖镁的核酸内切酶，并且确定了其在自然感染过程中的抗原性。MAG_5040 的 UGA 校正版本缺乏编码信号肽的区域，在大肠杆菌中作为 GST 融合蛋白表达。重组 GST-MAG_5040 表现出与典型的非特异性内切核酸酶和外切核酸酶相似的核酸酶活性，以 DNA 为首选底物，在 20mM MgCl2 存在下，温度范围为 37 至 45°C 时具有最佳活性。根据计算机分析，编码 MAG_5040 的基因的位置一致位于 ABC 转运蛋白的上游，在大多数属于人型支原体组的测序支原体中都是如此。在无乳链球菌中，MAG_5040 与 ABC 转运蛋白成分以及 MAG_5030 一起转录成多顺反子 RNA，MAG_5030 通过 3D 建模和同源搜索预测为糖溶质结合蛋白。在绵羊和山羊自然感染模型中，在感染后 9 个月检测到抗 MAG_5040 抗体。考虑到其酶活性，MAG_5040 可能在无乳链球菌进入宿主的生存中发挥关键作用，有助于宿主的致病性。MAG_5040 的鉴定为控制小反刍动物传染性无乳症提供了新的工具开发前景。
