New approaches to Study Pseudomonas-host interactions

描述(由申请人提供)：微生物病原体克服正常高度极化的宿主粘膜上皮屏障的能力是发病机制中早期和关键的一步。这对铜绿假单胞菌(PA)等机会性病原体尤其关键，PA是人类最致命的机会性病原体之一。然而，在上皮损伤和细胞极性丧失的情况下，PA可以有效地定植于粘膜表面，造成进一步的损伤，阻止损伤的上皮修复，并进行扩散。我们的长期目标是了解病原体，特别是PA是如何克服宿主上皮屏障而导致人类疾病的。我们的短期目标是了解PA如何劫持宿主信号通路进入细胞，以及细胞极性的破坏如何导致PA入侵。我们使用了一种新的正向遗传学方法，全基因组RNAi介导的基因失活，进行了有针对性的遗传筛选，以确定对细菌结合和内化重要的宿主蛋白。从这个屏幕上，我们已经鉴定了许多新的宿主基因，包括一个假定的受体(E-钙粘素)，适配器蛋白(Abl/Arg激酶和Crk)，细胞骨架的调节因子(CDC42，Rac和p21激活的蛋白激酶(Pak))，以及下游的效应因子(磷酸肌醇-3-磷酸激酶(PI3K)和Akt)。我们现在唯一准备确定的是，当PA进入哺乳动物细胞时，这些宿主分子是如何被破坏的，以及它是否与人类疾病有关。这些研究采用了新的遗传方法和最先进的细胞生物学，将全面剖析PA和宿主细胞上皮之间的相互作用。他们将确定细菌利用来致病的宿主因素。这些宿主细胞因子可能成为抗菌疗法发展的新靶点；因为药物针对宿主而不是细菌分子，与传统的抗微生物疗法相比，它们产生耐药性的可能性要小得多。

DESCRIPTION (provided by applicant): The ability of microbial pathogens to overcome the normally highly polarized host mucosal epithelial barrier is an early and critical step in pathogenesis. This is particularly critical for opportunistic pathogens such as Pseudomonas aeruginosa (PA), one of the most virulent opportunistic pathogens of man. In the setting of epithelial injury and loss of cell polarity, however, PA can effectively colonize the mucosal surfaces and cause further damage, prevent repair of the wounded epithelium, and disseminate. Our long term goals are to understand how pathogens in general, and PA in particular, overcome the host epithelial barrier to cause human disease. Our short term goals are to understand how PA hijacks host signaling pathways to enter into cells and how disruption of cell polarity predisposes to PA invasion. We have used a novel forward genetic approach, genome-wide RNAi-mediated gene inactivation, to carry out a targeted genetic screen to identify host proteins important for bacterial binding and internalization. From this screen, we have identified many new host genes required for entry of strain PAK, including a putative receptor (E-cadherin), adaptor proteins (Abl/arg kinase and Crk), regulators of the cytoskeleton (Cdc42, Rac, and p21-activated protein kinase (Pak)), and downstream effectors (phosphoinositol-3-phospho kinase (PI3K) and Akt). We are uniquely poised now to determine how these host molecules are subverted by PA when it enters mammalian cells and whether it is relevant to human disease. These studies, which employ novel genetic approaches and state-of-the-art cell biology, will comprehensively dissect the interactions between PA and host cell epithelium. They will identify host factors that the bacteria exploit to cause disease. These host cell factors may serve as novel targets for the development of anti-bacterial therapeutics; because the drug targets host but not bacterial molecules, they are much less likely to engender resistance compared to conventional anti-microbial therapies.