Kajfasz Jessica K, Rivera-Ramos Isamar, Scott-Anne Kathleen, Gregoire Stacy, Abranches Jacqueline, Lemos José A
Center for Oral Biology, University of Rochester Medical Center, Rochester, New York, USA.
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA.
J Bacteriol. 2015 Jul;197(13):2160-2170. doi: 10.1128/JB.00118-15. Epub 2015 Apr 20.
The SpxA1 and SpxA2 (formerly SpxA and SpxB) transcriptional regulators of Streptococcus mutans are members of a highly conserved family of proteins found in Firmicutes, and they were previously shown to activate oxidative stress responses. In this study, we showed that SpxA1 exerts substantial positive regulatory influence over oxidative stress genes following exposure to H2O2, while SpxA2 appears to have a secondary regulatory role. In vitro transcription (IVT) assays using purified SpxA1 and/or SpxA2 showed that SpxA1 and, less often, SpxA2 directly activate transcription of some of the major oxidative stress genes. Addition of equimolar concentrations of SpxA1 and SpxA2 to the IVT reactions neither enhanced transcription of the tested genes nor disrupted the dominant role of SpxA1. Substitution of a conserved glycine residue (G52) present in both Spx proteins by arginine (SpxG52R) resulted in strains that phenocopied the Δspx strains. Moreover, addition of purified SpxA1G52R completely failed to activate transcription of ahpC, sodA, and tpx, further confirming that the G52 residue is critical for Spx functionality.
Streptococcus mutans is a pathogen associated with the formation of dental caries in humans. Within the oral cavity, S. mutans routinely encounters oxidative stress. Our previous data revealed that two regulatory proteins, SpxA1 and SpxA2 (formerly SpxA and SpxB), bear high homology to the Spx regulator that has been characterized as a critical activator of oxidative stress genes in Bacillus subtilis. In this report, we prove that Spx proteins of S. mutans directly activate transcription of genes involved in the oxidative stress response, though SpxA1 appears to have a more dominant role than SpxA2. Therefore, the Spx regulators play a critical role in the ability of S. mutans to thrive within the oral cavity.
变形链球菌的SpxA1和SpxA2（以前称为SpxA和SpxB）转录调节因子是厚壁菌门中发现的高度保守蛋白质家族的成员，先前已证明它们可激活氧化应激反应。在本研究中，我们表明，暴露于H2O2后，SpxA1对氧化应激基因发挥了实质性的正向调节作用，而SpxA2似乎具有次要的调节作用。使用纯化的SpxA1和/或SpxA2进行的体外转录（IVT）分析表明，SpxA1以及较少情况下的SpxA2直接激活一些主要氧化应激基因的转录。向IVT反应中添加等摩尔浓度的SpxA1和SpxA2既没有增强测试基因的转录，也没有破坏SpxA1的主导作用。将两种Spx蛋白中存在的保守甘氨酸残基（G52）替换为精氨酸（SpxG52R）产生了表型与Δspx菌株相似的菌株。此外，添加纯化的SpxA1G52R完全无法激活ahpC、sodA和tpx的转录，进一步证实G52残基对Spx功能至关重要。
变形链球菌是一种与人类龋齿形成相关的病原体。在口腔中，变形链球菌经常会遇到氧化应激。我们之前的数据显示，两种调节蛋白SpxA1和SpxA2（以前称为SpxA和SpxB）与已被表征为枯草芽孢杆菌氧化应激基因关键激活剂的Spx调节因子具有高度同源性。在本报告中，我们证明变形链球菌的Spx蛋白直接激活参与氧化应激反应的基因的转录，尽管SpxA1似乎比SpxA2具有更主导的作用。因此，Spx调节因子在变形链球菌在口腔中生存的能力中起着关键作用。
