Transcriptional and Phenotypic Characterization of Novel Spx-Regulated Genes in Streptococcus mutans
Transcriptional and Phenotypic Characterization of Novel Spx-Regulated Genes in Streptococcus mutans
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DOI：
10.1371/journal.pone.0124969
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发表时间：
2015-04-23
期刊：
影响因子：
3.7
通讯作者：
Lemos, Jose A.
中科院分区：
文献类型：
--
作者：
Galvao, Lvia C. C.;Miller, James H.;Lemos, Jose A.
在口腔生物膜中，牙齿病原体变形链球菌遇到的两个主要环境挑战是酸和氧化应激。以前，我们表明，S。变异体转录调节因子SpxA 1和SpxA 2（分别以前称为SpxA和SpxB）通过激活经典氧化应激基因如dpr、nox、sodA和tpx的表达而参与应激存活。我们推断SpxA 1/A2控制下的一些未表征的基因可能参与氧化应激管理。因此，本研究的目的是利用Spx调控基因作为工具，以确定新的氧化应激基因在S。变异体实时定量PCR被用来评估10个Spx调控基因在H2 O2胁迫期间在亲本和Delta spx菌株中的响应。H2 O2诱导的基因的转录激活（10个中的8个）强烈依赖于SpxA 1，在较小程度上，SpxA 2。体外转录试验表明，一个或两个Spx蛋白直接调节这些基因中的三个。编码FeoB亚铁通透酶的基因被H2 O2轻微抑制，但在缺乏SpxA 1的菌株中组成型诱导。选择9个基因进行下游突变分析，但失活的smu 127，编码亚基的乙偶姻脱氢酶显然是致命的。在体外和体内表征的可行的突变体表明，除了转录激活的还原和抗氧化途径，Spx通过调节细胞内游离铁的可用性在铁稳态中发挥重要作用。特别是，失活的基因编码的Fe-S生物合成SUF系统和以前的特点铁结合蛋白DPR导致在不同的氧化应激条件下生长受损，增加对铁的敏感性和降低感染性的大鼠。这些结果作为一个入口进入新的基因和途径，使S。变形菌来科普氧化应激。
In oral biofilms, two of the major environmental challenges encountered by the dental pathogen Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the S. mutans transcriptional regulators SpxA1 and SpxA2 (formerly SpxA and SpxB, respectively) are involved in stress survival by activating the expression of classic oxidative stress genes such as dpr, nox, sodA and tpx. We reasoned that some of the uncharacterized genes under SpxA1/A2 control are potentially involved in oxidative stress management. Therefore, the goal of this study was to use Spx-regulated genes as a tool to identify novel oxidative stress genes in S. mutans. Quantitative real-time PCR was used to evaluate the responses of ten Spx-regulated genes during H2O2 stress in the parent and Delta spx strains. Transcription activation of the H2O2-induced genes (8 out of 10) was strongly dependent on SpxA1 and, to a lesser extent, SpxA2. In vitro transcription assays revealed that one or both Spx proteins directly regulate three of these genes. The gene encoding the FeoB ferrous permease was slightly repressed by H2O2 but constitutively induced in strains lacking SpxA1. Nine genes were selected for downstream mutational analysis but inactivation of smu127, encoding a subunit of the acetoin dehydrogenase was apparently lethal. In vitro and in vivo characterization of the viable mutants indicated that, in addition to the transcriptional activation of reducing and antioxidant pathways, Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron. In particular, inactivation of the genes encoding the Fe-S biogenesis SUF system and the previously characterized iron-binding protein Dpr resulted in impaired growth under different oxidative stress conditions, increased sensitivity to iron and lower infectivity in rats. These results serve as an entryway into the characterization of novel genes and pathways that allow S. mutans to cope with oxidative stress.