Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System
Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System
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DOI：
10.1128/jb.00049-16
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发表时间：
2016-05-01
影响因子：
3.2
通讯作者：
Yahr, Timothy L.
中科院分区：
文献类型：
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作者：
Marsden, Anne E.;Intile, Peter J.;Yahr, Timothy L.
铜绿假单胞菌环腺苷酸（cAMP）-Vfr系统（CVS）是毒力基因表达的全局调节因子。调节靶标包括IV型皮利、分泌蛋白酶和III型分泌系统（T3 SS）。CVS调节T3 SS基因表达的机制尚不清楚。单细胞表达研究先前发现，在诱导条件下，群体中只有一部分细胞表达T3 SS，这是一种称为双稳态的特性。我们现在报告说，双稳态改变vfr突变体，其中一个基本上较小的分数的细胞表达T3 SS相对于亲本菌株。由于双稳态通常涉及正反馈回路，我们测试的假设，毒力因子调节因子（VFR）调节exsA的表达。ExsA是T3 SS基因表达的中心调节因子，并自动调节其自身的表达。虽然exsA是exsCEBA多顺反子mRNA的最后一个基因，但我们证明Vfr直接激活位于exsA上游的第二个启动子（P-exsA）的exsA转录。P-exsA启动子活性完全依赖于Vfr。电泳迁移率变动分析表明，直接结合的VFR的P-exsA启动子探针，和DNA酶I足迹揭示了一个领域的保护，符合一个假定的VFR共识结合位点。该位点的突变破坏了Vfr结合和P-exsA启动子活性。我们的结论是，Vfr有助于T3 SS基因的表达，通过激活P-exsA启动子，这是内部的先前表征exsCEBA operon.IMPORTANCEVfr是一个cAMP依赖的DNA结合蛋白，作为一个全球调节器的铜绿假单胞菌的毒力基因的表达。Vfr的调节允许相关毒力功能的协调产生，例如IV型皮利和III型分泌，其分别是粘附和中毒宿主细胞所需的。虽然Vfr调控的分子机制已被确定为许多靶基因，Vfr和T3 SS基因表达之间的直接联系尚未建立。在本研究中，我们报告说，Vfr直接控制exsA转录，T3 SS基因表达的主调节器，从一个新发现的启动子位于紧邻exsA的上游。
The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA. ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (P-exsA) located immediately upstream of exsA. P-exsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a P-exsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and P-exsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the P-exsA promoter, which is internal to the previously characterized exsCEBA operon.IMPORTANCEVfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa. Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA.