Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America.
Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America.
PLoS One. 2014 Apr 21;9(4):e95574. doi: 10.1371/journal.pone.0095574. eCollection 2014.
Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc) enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2) that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET) assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.
金黄色葡萄球菌是一种重要的细菌病原体，可引起多种急性和慢性感染。最近的研究表明，分泌的核酸酶（Nuc）是多种感染模型中的一种毒力因子，而体内表达 nuc 有助于开发基于 Nuc 激活探针的感染成像方法。有趣的是，金黄色葡萄球菌株编码第二种核酸酶（Nuc2），但其受到的关注有限。随着对细菌核酸酶的兴趣日益增加，我们试图通过定位、表达和生化研究更详细地描述 Nuc2。使用 Nuc2-GFP 和 Nuc2-PhoA 融合蛋白的荧光显微镜和碱性磷酸酶定位方法，分别证明 Nuc2 与细胞膜结合，C 端面向细胞外环境，表明它是一种信号锚定的 II 型膜蛋白。使用荧光共振能量转移（FRET）测定法在金黄色葡萄球菌细胞表面上可检测到 Nuc2 酶活性，并且在时间过程中，nuc2 转录和酶活性在对数早期生长时达到峰值，并在静止期下降。在金黄色葡萄球菌肌炎的小鼠模型中，使用激活探针在体内检测到 Nuc2 活性，表明在感染过程中产生了 Nuc2。为了评估 Nuc2 的生化特性，纯化了该蛋白，发现它可以切割单链和双链 DNA，并且它表现出热稳定性和钙依赖性，与 Nuc 的特性相似。纯化的 Nuc2 可在体外防止生物膜形成，并在分散实验中适度降低生物量。总的来说，我们的研究结果证实金黄色葡萄球菌编码第二种表面附着且有功能的 DNA 酶，该酶在感染过程中表达，并显示出与分泌型 Nuc 酶相似的生化特性。
