Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.
Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.
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DOI：
10.1371/journal.pone.0095574
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Horswill AR
中科院分区：
文献类型：
--
作者：
Kiedrowski MR;Crosby HA;Hernandez FJ;Malone CL;McNamara JO 2nd;Horswill AR
金黄色葡萄球菌是一种引起各种急性和慢性感染的主要细菌病原体。最近，它已被证明，分泌的核酸酶（Nuc）酶是一种毒力因子，在多种模式的感染，和在体内表达的nuc促进了发展的感染成像方法的基础上Nuc激活探针。有趣的是，S.金黄色葡萄球菌菌株编码的第二种核酸酶（Nuc 2）受到了有限的关注。随着人们对细菌核酸酶的兴趣越来越大，我们试图通过定位、表达和生物化学研究更详细地描述Nuc 2。分别使用Nuc 2-GFP和Nuc 2-PhoA融合的荧光显微镜和碱性磷酸酶定位方法证明，Nuc 2与面向细胞外环境的C-末端膜结合，表明它是信号锚定的II型膜蛋白。在S.金黄色葡萄球菌细胞表面的荧光共振能量转移（FRET）测定，并在时间过程中，nuc 2转录和酶活性达到高峰，在对数生长早期，并在稳定期下降。利用S.在金黄色葡萄球菌化脓性肌炎中，在nuc突变株中用可活化探针在体内检测Nuc 2活性，表明Nuc 2在感染期间产生。为了评估Nuc 2的生物化学性质，对该蛋白进行纯化，发现其可切割单链和双链DNA，并且其表现出热稳定性和钙依赖性，与Nuc的性质平行。纯化的Nuc 2在体外防止生物膜形成，并在扩散实验中适度降低生物量。总之，我们的研究结果证实，S。金黄色葡萄球菌编码第二种表面附着的功能性DNA酶，其在感染期间表达，并显示出与分泌的Nuc酶相似的生化性质。
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.
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影响因子：
4.4
作者：
Heun, Magnus;Binnenkade, Lucas;Thormann, Kai M.
通讯作者：
Thormann, Kai M.
影响因子：
4
作者：
Hernandez, Frank J.;Stockdale, Katie R.;McNamara, James O., II
通讯作者：
McNamara, James O., II
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
3.2
作者：
Beenken, KE;Dunman, PM;Smeltzer, MS
通讯作者：
Smeltzer, MS
影响因子：
3.1
作者：
GILLASPY, AF;HICKMON, SG;SMELTZER, MS
通讯作者：
SMELTZER, MS