Post-transcriptional regulation of Pseudomonas virulence gene expression

描述（由申请方提供）：许多铜绿假单胞菌毒力决定因子在急性或慢性定植期优先表达。例如，III型分泌（T3 SS）和cAMP/Vfr信号传导（CVS）系统的表达对于急性动物感染模型中的毒力是重要的。然而，这些相同的因子在从患有慢性铜绿假单胞菌感染的囊性纤维化（CF）患者获得的分离物中不表达。至少有两种全局调节途径（RsmA和MucA/AlgU）反向调节T3 SS/CVS和慢性毒力基因表达。RsmA通路通过生物膜形成（常见于CF气道）反向调节T3 SS/CVS基因表达，并且依赖于RsmA，一种在转录后水平调节基因表达的RNA结合蛋白。MucA/AlgU途径激活类粘蛋白转化（CF分离株的共同特征）但抑制T3 SS/CVS基因表达。我们的初步数据表明，RsmA和AlgU/MucA调节途径在转录后水平调节ExsA（T3 SS的主要转录调节因子）和Vfr表达。这些研究的长期目标是通过解决以下具体目标，使用T3 SS和CVS作为模型系统来定义管理急性向慢性过渡的调节机制。 目标1。确定RsmA如何在转录后水平促进ExsA和Vfr表达。我们的初步数据表明，纯化的RsmA直接结合到5'非翻译区的vfr和exsA的mRNA，并刺激Vfr表达在体外翻译测定。基于这些数据，我们将测试一种新的翻译去阻遏模型，其中RsmA结合缓解exsA和vfr mRNA内的二级结构，隔离核糖体结合位点或以其他方式抑制翻译。 目标二。确定MucA/AlgU系统如何改变游离RsmA的可用性以控制ExsA和CVS表达。我们的数据表明，RsmA，RsmY和RsmZ的表达显着升高的mucA突变体。在此目的中提出的实验将检验MucA/AlgU系统通过降低细胞中游离RsmA的浓度来控制Vfr和ExsA表达的假设。 目标3。确定RsmB活性是如何控制的，以及RsmB如何在转录后水平促进ExsA和Vfr表达。虽然质粒表达的RsmB恢复ExsA和Vfr表达的rsmA，rsmB双突变体，我们发现，纯化的RsmB不结合相同的VFR和exsA mRNA探针结合RsmA。然而，纯化的RsmA和RsmB确实以相似的亲和力与对照探针结合，表明纯化的RsmB是有活性的。为了确定RsmB如何促进ExsA和Vfr表达，我们将绘制RsmB反应区，并询问RsmB是否在体内与exsA和vfr mRNA结合。

DESCRIPTION (provided by applicant): Many Pseudomonas aeruginosa virulence determinants are preferentially expressed during the acute or chronic phases of colonization. For instance, expression of the type III secretion (T3SS) and the cAMP/Vfr signaling (CVS) systems is important for virulence in acute animal infection models. These same factors, however, are not expressed in isolates obtained from cystic fibrosis (CF) patients with chronic P. aeruginosa infections. At least two global regulatory pathways (RsmA and MucA/AlgU) inversely regulate T3SS/CVS and chronic virulence gene expression. The RsmA pathway inversely regulates T3SS/CVS gene expression with biofilm formation (common in the CF airways), and is dependent upon RsmA, an RNA binding protein that regulates gene expression at the post-transcriptional level. The MucA/AlgU pathway activates mucoid conversion (a common trait of CF isolates) but inhibits T3SS/CVS gene expression. Our preliminary data demonstrate that the RsmA and AlgU/MucA regulatory pathways modulate ExsA (the primary transcriptional regulator of the T3SS) and Vfr expression at the post-transcriptional level. The long-term goal of these studies is to define regulatory mechanisms that govern the acute to chronic transition using the T3SS and CVS as model systems by addressing the following specific aims. Aim 1. Determine how RsmA promotes ExsA and Vfr expression at the post-transcriptional level. Our preliminary data indicate that purified RsmA binds directly to the 5' untranslated regions of the vfr and exsA mRNAs, and stimulates Vfr expression in an in vitro translation assay. Based on these data we will test a novel translational derepression model whereby RsmA binding relieves secondary structures within the exsA and vfr mRNAs that sequester the ribosome binding site or otherwise inhibit translation. Aim 2. Determine how the MucA/AlgU system alters the availability of free RsmA to control ExsA and CVS expression. Our data indicate that the expression of RsmA, RsmY, and RsmZ are significantly elevated in the mucA mutant. The experiments proposed in this aim will test the hypothesis that the MucA/AlgU system controls Vfr and ExsA expression by reducing the concentration of free RsmA in cells. Aim 3. Determine how RsmB activity is controlled and how RsmB promotes ExsA and Vfr expression at the post-transcriptional level. Although plasmid-expressed RsmB restores ExsA and Vfr expression in an rsmA,rsmB double mutant, we find that purified RsmB does not bind to the same vfr and exsA mRNA probes bound by RsmA. Purified RsmA and RsmB, however, do bind with similar affinity to a control probe indicating that purified RsmB is active. To determine how RsmB promotes ExsA and Vfr expression we will map the RsmB-responsive region and ask whether RsmB binds to the exsA and vfr mRNAs in vivo.