ability of microbial pathogens to overcome the normally highly polarized host mucosal ..epithelial'.barrier is an early and critical step in microbial pathogenesis. This is particularly true 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, 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 from either the apical (AP) or basolateral (BL) surface of the mucosal barrier and how disruption of cell polarity predisposes to PA invasion. We have used model tissue culture systems to'uncover host glycoconjugate receptors and cell signaling systems that are subverted by the pathogen and are critical to invasion. Our collaborators have developed novel methods to more closely model the epithelial barrier by growing human lung cells in 2D and 3 D cultures. We will apply these advances to study the interaction of PA with the AP and BL surfaces of polarized epithelium. Specifically, we will (1) We will elucidate the signaling pathways involved in the interactions of PA and polarized host cells through N-glycoproteins at the AP surface and heparan sulfate proteoglycans on the BL surface. 2. We will examine the characteristics of binding, entry, signaling, and cytotoxicity of PA to MDCK cells, primary human alveolar type II epithelial cellsand human bronchial epithelial cells (i6HBE14o-) grown as cysts in 3D cultures. 3. We will test the hypothesis that sulf-1 and/or sulf-2, key enzymes that regulate cell surface HSPG 6-O sulfation, affect PA interactions with the mucosal barrier in normal or wounded epithelium and in animal models of acute infection. We will also test whether PA interferes with sulfatase and Wnt regulated wound repair. Together, these studies will comprehensively dissect the interactions between PA and the host mucosal barrier to identify host molecules that the bacteria exploit to cause disease. This work examines how an important human disease causing bacteria gets across the skin and other mucosal barriers to cause disease. It has the potential to identify new therapeutics to treat this increasingly antibiotic resistant organism.