Gene Expression and Pathogenicity of P.aeruginosa

描述（由申请人提供）： 我们建议进行一项转化研究，包括临床和基础科学家，以检验特定假单胞菌毒力产物决定肺部感染是否发生的假设。拟议实验的结果将用于开发诊断工具，可以识别患者中的有毒假单胞菌。研究结果还将确定与肺损伤有关的关键细菌基因产物。本申请仔细剖析了细菌毒力和肺损伤之间的关联，从而可以提出未来在患者中的机制研究。如果没有拟议调查的数据，我们就没有继续进行调查的信息。我们已经从另一个基金会的一组插管、机械通气患者中获得了假单胞菌菌株。我们的第一个目标是对从2个不同的患者人群中获得的假单胞菌菌株进行基因组分析;具有肺部感染迹象的插管通气患者已经扩散，而插管通气患者没有疾病迹象。使用微阵列分析，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，并产生针对这些基因的共有引物。然后，我们将在300例插管、通气患者的前瞻性调查中利用这些共有引物，以确认定植与感染的插管、通气患者的遗传差异的患病率。第二个目的是比较从定殖患者获得的假单胞菌菌株的体内毒力基因表达与从患有假单胞菌肺感染的患者获得的菌株的表达谱。将使用实时PCR进行体内细菌基因表达的分析。所有这些实验的结果将使我们能够证明（或反驳），在感染患者的假单胞菌菌株和定植患者的假单胞菌菌株之间存在不同的基因或不同的毒素基因表达模式。在最后的目标中，我们将评估III型毒素基因在体内的相互作用。通过利用具有单独或组合添加的每种毒素基因的同基因细菌菌株（PA 103），我们将能够确定每种III型毒素以及我们发现在前两个目标中具有重要意义的其他基因的作用。

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.