An unconventional RNA-based thermosensor within the 5’ UTR of Staphylococcus aureus cidA

Staphylococcus aureus is a Gram-positive bacterial pathogen of global concern and a leading cause of bacterial infections worldwide. Asymptomatic carriage of S. aureus on the skin and in the anterior nares is common and recognized as a predisposing factor to invasive infection. Transition of S. aureus from the carriage state to that of invasive infection is often accompanied by a temperature upshift from approximately 33°C to 37°C. Such a temperature shift is known in other pathogens to influence gene expression, often resulting in increased production of factors that promote survival or virulence within the host. One mechanism by which bacteria modulate gene expression in response to temperature is by the regulatory activity of RNA-based thermosensors, cis-acting riboregulators that control translation efficiency. This study was designed to identify and characterize RNA-based thermosensors in S. aureus. Initially predicted by in silico analyses of the S. aureus USA300 genome, reporter-based gene expression analyses and site-specific mutagenesis were performed to demonstrate the presence of a functional thermosensor within the 5’ UTR of cidA, a gene implicated in biofilm formation and survival of the pathogen. The nucleic sequence composing the identified thermosensor are sufficient to confer temperature-dependent post-transcriptional regulation, and activity is predictably altered by the introduction of site-specific mutations designed to stabilize or destabilize the structure within the identified thermosensor. The identified regulator is functional in both the native bacterial host S. aureus and in the distally related species Escherichia coli, suggesting that its regulatory activity is independent of host-specific factors. Interestingly, unlike the majority of bacterial RNA-based thermosensors characterized to date, the cidA thermosensor facilitates increased target gene expression at lower temperatures. In addition to the characterization of the first RNA-based thermosensor in the significant pathogen S. aureus, it highlights the diversity of function within this important class of ribo-regulators.金黄色葡萄球菌（Staphylococcus aureus）是一类备受全球关注的革兰氏阳性致病菌，也是全球细菌性感染的首要诱因。该菌在皮肤与前鼻孔的无症状定植十分普遍，且已被证实是引发侵袭性感染的易感因素。金黄色葡萄球菌从定植状态转为侵袭性感染状态时，通常伴随温度从约33°C升高至37°C。在其他致病菌中，这类温度变化已被证实会影响基因表达，往往会增加促进宿主内生存或毒力的因子的产生。细菌响应温度调控基因表达的机制之一，是基于RNA的温度传感器（RNA-based thermosensors）的调控活性——这类顺式作用核糖调节因子（cis-acting riboregulators）可控制翻译效率。本研究旨在鉴定并解析金黄色葡萄球菌中的基于RNA的温度传感器。研究最初通过对金黄色葡萄球菌USA300基因组的计算机模拟（in silico）分析进行预测，随后通过基于报告基因的基因表达分析与定点诱变（site-specific mutagenesis）实验，证实了cidA基因5’非翻译区（5’ UTR）内存在功能性温度传感器；cidA基因与该致病菌的生物膜形成及生存密切相关。构成该鉴定得到的温度传感器的核酸序列足以赋予温度依赖性的转录后调控（post-transcriptional regulation）功能，且通过引入旨在稳定或破坏该温度传感器内部结构的定点突变，可预期改变其调控活性。该调控因子在天然宿主金黄色葡萄球菌以及亲缘关系较远的大肠杆菌（Escherichia coli）中均能发挥功能，这提示其调控活性不依赖宿主特异性因子。值得注意的是，与目前已表征的多数细菌基于RNA的温度传感器不同，cidA温度传感器可在较低温度下促进靶基因的表达上调。本研究不仅鉴定了首个在重要致病菌金黄色葡萄球菌中存在的基于RNA的温度传感器，还凸显了这类重要核糖调节因子家族的功能多样性。