Post-transcriptional regulation of Pseudomonas virulence gene expression

描述(由申请人提供)：许多铜绿假单胞菌毒力决定因素在定植的急性或慢性阶段优先表达。例如，III型分泌物(T3SS)和cAMP/Vfr信号系统(CVS)的表达对于急性动物感染模型的毒力是重要的。然而，这些相同的因子在囊性纤维化(CF)患者合并慢性铜绿假单胞菌感染的分离株中不表达。至少有两条全球调控通路(RSMA和MUTA/ALGU)反向调节T3SS/CVS和慢性毒力基因的表达。RSMA途径通过生物膜的形成反向调节T3SS/CVS基因的表达(在CF呼吸道中常见)，并依赖于RSMA，RSMA是一种RNA结合蛋白，在转录后水平调节基因的表达。粘液A/ALGU途径激活粘液转化(这是CF分离株的共同特征)，但抑制T3SS/CVS基因的表达。我们的初步数据表明，RSMA和ALGU/MUTA调节通路在转录后水平调控ExsA(T3SS的主要转录调节因子)和Vfr的表达。这些研究的长期目标是通过解决以下具体目标，以T3SS和CVS作为模型系统，确定管理急性到慢性转变的调控机制。目的1.确定RSMA如何在转录后水平促进ExsA和Vfr的表达。我们的初步数据表明，纯化的RSMA直接与Vfr和exsA mRNAs的5‘非翻译区结合，并在体外翻译实验中刺激Vfr的表达。基于这些数据，我们将测试一种新的翻译去抑制模型，通过该模型，RSMA结合解除了exsA和VFR mRNAs中的二级结构，这些结构隔离了核糖体结合部位或以其他方式抑制了翻译。目的2.确定MUSA/ALGU系统如何改变游离RSMA的可用性来控制ExsA和CVS的表达。我们的数据表明，在粘蛋白A突变体中，RSMA、RsmY和RsmZ的表达显著升高。本实验旨在验证粘液A/ALGU系统通过降低细胞内游离RSMA浓度来控制Vfr和ExsA表达的假说。目的3.确定RSMB活性如何被控制，以及RSMB如何在转录后水平促进ExsA和Vfr的表达。尽管表达的RSMB在RSMA、RSMB双突变体中恢复了ExsA和Vfr的表达，但我们发现纯化的RSMB不能与RSMA结合的Vfr和exsA mRNA探针结合。然而，纯化的RSMA和RSMB确实与对照探针具有类似的亲和力，表明纯化的RSMB是活性的。为了确定RSMB如何促进ExsA和Vfr的表达，我们将绘制RSMB响应区的图谱，并询问RSMB是否与体内的exsA和Vfr mRNAs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.