New approaches to study Pseudomonas-host interactions

描述（由申请人提供）：病原体突破高度极化宿主粘膜上皮屏障的能力是微生物感染发病机制的早期和关键步骤。这一事件对铜绿假单胞菌（PA）等机会致病菌尤其重要，这是人类最致命的机会致病菌之一。我们的长期目标是了解一般的病原体，特别是PA如何克服宿主上皮屏障导致人类疾病。PA结合到极化上皮细胞的顶端（AP）表面，引发宿主细胞的根本变化，允许细菌内化。在此期间，我们发现PA诱导AP表面形态发生显著的局部变化，形成由基底外侧（BL）成分组成的膜状突起。这个突出物很可能是细菌进入的通道。这种AP膜局部特性的深刻转变是已知的PA对上皮细胞单层影响的第一个形态学里程碑。根据我们的数据，我们假设PA可以利用粘膜屏障上皮极性的小缺陷来启动一系列事件，导致正反馈循环，诱导AP膜转化为BL膜的显着形态变化。形成一个具有新生AJ特征的突起，作为细菌进入的通道。通过操纵上皮细胞极性，PA能够增强其细菌结合和进入，可能是通过增加宿主细胞受体的呈递或其他促进内化的因素。我们的目标是：(1)确定TJ和AJ的哪些关键调节和结构成分与突出有关。(2)确定突出物形成与细菌进入之间的时间和功能关系。(3)进行机制研究，揭示PA如何劫持宿主细胞囊泡运输，将这些连接和BL成分重定向到AP表面。(4)确定引起突出的细菌决定因素。总之，这些研究将全面剖析PA与宿主细胞上皮之间的相互作用。他们将确定细菌利用的致病宿主因素。利用病原体来研究哺乳动物生物学的基本过程，如上皮极性的建立和维持，除了发病机制外，还对包括癌症和发育生物学在内的许多领域具有广泛的影响。此外，这些宿主细胞因子可能成为开发抗菌治疗药物的新靶点；由于这种药物针对的是宿主而不是细菌分子，因此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 a pathogen to breach the highly polarized host mucosal epithelial barrier is an early and critical step in the pathogenesis of microbial infections. This event is particularly crucial for opportunistic pathogens such as Pseudomonas aeruginosa (PA), one of the most virulent opportunistic pathogens of man. Our long-term goals are to understand how pathogens in general, and PA in particular, overcome the host epithelial barrier to cause human disease. PA binds to the apical (AP) surface of polarized epithelial cells and triggers fundamental changes in the host cell that allow bacterial internalization. During this granting period, we have discovered that PA induces a remarkable local change in the morphology of the AP surface to form a membranous protrusion composed of basolateral (BL) constituents. This protrusion is the likely conduit for bacterial entry. This profound shift in the local identity of the AP membrane is the first known morphologic landmark of the effect of PA on epithelial cell monolayers. Based on our data, we hypothesize that PA can exploit small defects in epithelial polarity at the mucosal barrier to initiative a series of events that result in a positive feedback loop induces a remarkable morphologic change in which AP membrane is transformed into BL membrane. A protrusion, with characteristics of a nascent AJ, is formed that functions as a conduit for bacterial entry. By manipulating epithelial cell polarity, PA is able to enhance its bacterial binding and entry, possibly by increasing the presentation of host cell receptors or other factors that promote internalization. Our aims are to (1) Determine which key regulatory and structural components of TJ and AJ are recruited to the protrusion. (2) Determine the temporal and functional relationship between protrusion formation and bacterial entry. (3) Perform mechanistic studies to uncover how PA hijacks host cell vesicular trafficking to redirect these junctional and BL components to the AP surface. (4) Identify the bacterial determinants of protrusion formation. Together, these studies will comprehensively dissect the interactions between PA and host cell epithelium. They will identify host factors that the bacteria exploit to cause disease. The use of pathogens to study fundamental processes in mammalian biology, such as the establishment and the maintenance of epithelial polarity, has broad ramifications for many fields in addition to pathogenesis, including cancer and developmental biology. Furthermore, 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.