Gene Expression and Pathogenicity of P.aeruginosa

描述(由申请人提供)： 我们正在提议一项包括临床和基础科学家在内的翻译研究，以检验特定的假单胞菌毒力产品决定是否发生肺部感染的假设。拟议实验的结果将被用于开发诊断工具，可以识别患者中的毒力假单胞菌。结果还将确定与肺损伤有关的关键细菌基因产物。目前的应用仔细分析了细菌毒力和肺损伤之间的联系，以便对患者的未来机制研究提出建议。如果没有拟议调查的数据，我们就没有继续进行的信息。我们已经有了从另一笔赠款中获得的一批插管、机械通气患者的假单胞菌菌株。我们的第一个目标是对从两个不同的患者群体中获得的假单胞菌菌株进行基因组分析；这两个患者群体分别是有肺部感染迹象的插管患者和没有疾病迹象的插管患者和呼吸机患者。利用基因芯片分析，我们将确定在这两个患者组中发现的假单胞菌菌株之间的显著差异基因，并针对这些基因产生一致的引物。然后，我们将在一项对300名插管、机械通气患者的前瞻性研究中使用这些公认的引物，以确认定植的和感染的插管、机械通气患者的遗传差异的发生率。第二个目的是比较从定植患者体内获得的假单胞菌毒力基因表达与从假单胞菌肺部感染患者获得的菌株的表达谱。利用实时荧光定量聚合酶链式反应分析细菌体内基因表达。所有这些实验的结果将使我们能够证明(或反驳)感染患者的假单胞菌菌株与定植患者之间存在不同的基因或毒素基因表达模式。在最终目标中，我们将利用一个同基因的细菌菌株(PA103)来评估III型毒素基因在体内的相互作用，该菌株将每个毒素基因单独或组合添加，我们将能够定义每个III型毒素以及我们在前两个目标中发现的其他有意义的基因的影响。

DESCRIPTION (provided by applicant): We are proposing a translational investigation, including clinical as well as basic scientists, to test the hypothesis that specific Pseudomonas virulence products determine whether lung infection occurs. The results from the proposed experiments will be used to develop diagnostic tools that can identify virulent Pseudomonas in patients. The results will also identify critical bacterial gene products involved in lung injury. The present application carefully dissects associations between bacterial virulence and lung injury so that future mechanistic investigations in patients can be proposed. Without the data from the proposed investigations, we do not have the information to proceed. We already have Pseudomonas strains obtained from a cohort of intubated, mechanically ventilated patients from another grant. Our first aim is to perform a genomic analysis of Pseudomonas strains obtained from 2 distinct patient populations; intubated, ventilated patients with signs of lung infections that have disseminated and intubated, ventilated patients who have no sign of disease. Using microarray analysis, we will determine the significantly different genes between the Pseudomonas strains found in these two patient groups and produce consensus primers to these genes. We will then utilize these consensus primers in a prospective investigation of 300 intubated, ventilated patients to confirm the prevalence of the genetic differences in colonized vs infected intubated, ventilated patients. The second aim is to compare the in vivo virulence gene expression of Pseudomonas strains obtained from colonized patients to the expression profile of the strains obtained from patients who have Pseudomonas lung infections Analysis of bacterial gene expression in vivo will be done using real-time PCR. The results of all these experiments will allow us to prove (or disprove) that there are different genes or different patterns of toxin gene expression between the Pseudomonas strains infecting patients and those colonizing patients. In the final aim, we will evaluate the interaction of type III toxin genes in vivo By utilizing an isogenic bacterial strain (PA103) that has each of the toxin genes added alone or in combination, we will be able to define the effects of each of the type III toxins as well as other genes we find of significance in the first two aims.