Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Anal Biochem. 2013 Dec 1;443(1):81-7. doi: 10.1016/j.ab.2013.08.018. Epub 2013 Aug 29.
The mazEFSa toxin-antitoxin (TA) system is ubiquitous in clinical isolates of Staphylococcus aureus, yet its physiological role is unclear. MazFSa is a sequence-specific endoribonuclease that inhibits the growth of S. aureus and Escherichia coli on ectopic overexpression. MazFSa preferentially cleaves RNA at UACAU sites, which are overrepresented in genes encoding pathogenicity factors. The exploitation of the inherent toxicity of MazFSa by artificial toxin activation has been proposed as an antibacterial strategy; however, enzymatic activity of endogenous MazFSa has never been detected, and tools for such analyses are lacking. Here we detail methods for detection of the ribonuclease activity of MazFSa, including a continuous fluorometric assay and a gel-based cleavage assay. Importantly, these methods allowed for the first detection of endogenous MazFSa enzymatic activity in S. aureus lysate. These robust and sensitive assays provide a toolkit for the identification, analysis, and validation of stressors that induce MazF enzymatic activity and should assist in the discovery of artificial activators of the mazEFSa TA system.
MazEFSa 毒素-抗毒素（TA）系统在金黄色葡萄球菌的临床分离株中普遍存在，但它的生理作用尚不清楚。MazFSa 是一种序列特异性内切核糖核酸酶，可抑制金黄色葡萄球菌和大肠杆菌在异位过表达时的生长。MazFSa 优先在 UACAU 位点切割 RNA，这些位点在编码致病性因子的基因中过度表达。通过人工毒素激活利用 MazFSa 的固有毒性已被提议作为一种抗菌策略；然而，从未检测到内源性 MazFSa 的酶活性，并且缺乏用于此类分析的工具。在这里，我们详细介绍了检测 MazFSa 核糖核酸酶活性的方法，包括连续荧光测定法和基于凝胶的切割测定法。重要的是，这些方法首次检测到了金黄色葡萄球菌裂解物中内源性 MazFSa 的酶活性。这些强大而敏感的测定法为鉴定、分析和验证诱导 MazF 酶活性的应激源提供了一个工具包，应该有助于发现 mazEFSa TA 系统的人工激活剂。
