CbpA: a Polarly Localized Novel Cyclic AMP-Binding Protein in Pseudomonas aeruginosa
CbpA: a Polarly Localized Novel Cyclic AMP-Binding Protein in Pseudomonas aeruginosa
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DOI：
10.1128/jb.00970-09
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发表时间：
2009-12-01
影响因子：
3.2
通讯作者：
Engel, Joanne N.
中科院分区：
文献类型：
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作者：
Endoh, Takayuki;Engel, Joanne N.
在铜绿假单胞菌中，环磷酸腺苷(CAMP)信号调节数百个编码多种毒力因子的基因的转录，包括II型分泌系统(T2SS)和III型分泌系统(T3SS)及其相关毒素、IV型菌毛(TFP)和鞭毛。Vfr是一种依赖cAMP的转录调节因子，与大肠杆菌分解代谢抑制蛋白同源，被认为是调节这些重要毒力决定因素的主要cAMP结合蛋白。利用生物信息学方法，我们在铜绿假单胞菌PAO1中发现了一个额外编码cAMP结合蛋白的基因(PA4704)，我们称之为CbpA，结构模拟预测CbpA由一个C端cAMP结合(CAP)结构域和一个N端简并CAP结构域组成，在结构上类似于真核蛋白激酶A调节亚基。我们发现CbpA通过其C端的CAP结构域与cAMP偶联的琼脂糖结合。使用体外胰酶保护实验，我们证明了cbpA在cAMP结合后发生了构象变化。报告基因分析和凝胶迁移率改变分析确定了CBPA启动子和Vfr结合位点，这是Vfr依赖的转录所必需的。尽管CbpA受到Vfr的高度调控，但CBPA的缺失并不影响已知的Vfr依赖功能，包括T2SS、T3SS、鞭毛或TFP依赖的运动性、急性肺炎小鼠模型的毒力或蛋白质表达谱。出乎意料的是，CbpA-绿色荧光蛋白被发现以cAMP依赖的方式定位于有鞭毛的旧细胞极点。这些结果表明，CbpA的极性定位可能对其功能有重要作用。
In Pseudomonas aeruginosa, cyclic AMP (cAMP) signaling regulates the transcription of hundreds of genes encoding diverse virulence factors, including the type II secretion system (T2SS) and type III secretion system (T3SS) and their associated toxins, type IV pili (TFP), and flagella. Vfr, a cAMP-dependent transcriptional regulator that is homologous to the Escherichia coli catabolite repressor protein, is thought to be the major cAMP-binding protein that regulates these important virulence determinants. Using a bioinformatic approach, we have identified a gene (PA4704) encoding an additional putative cAMP-binding protein in P. aeruginosa PAO1, which we herein refer to as CbpA, for cAMP-binding protein A. Structural modeling predicts that CbpA is composed of a C-terminal cAMP-binding (CAP) domain and an N-terminal degenerate CAP domain and is structurally similar to eukaryotic protein kinase A regulatory subunits. We show that CbpA binds to cAMP-conjugated agarose via its C-terminal CAP domain. Using in vitro trypsin protection assays, we demonstrate that CbpA undergoes a conformational change upon cAMP binding. Reporter gene assays and electrophoresis mobility shift assays defined the cbpA promoter and a Vfr-binding site that are necessary for Vfr-dependent transcription. Although CbpA is highly regulated by Vfr, deletion of cbpA did not affect known Vfr-dependent functions, including the T2SS, the T3SS, flagellum-or TFP-dependent motility, virulence in a mouse model of acute pneumonia, or protein expression profiles. Unexpectedly, CbpA-green fluorescent protein was found to be localized to the flagellated old cell pole in a cAMP-dependent manner. These results suggest that polar localization of CbpA may be important for its function.