PROJECT SUMMARY Biofilm formation represents a protective mode of growth that allows microorganisms to survive in hostile environments and disperse cells to colonize new niches under desirable conditions. When established at undesired locations, these microbial communities are of great concern clinically and industrially, in part due to their ability to tolerate a wide range of treatment and eradication strategies. A better understanding of the genetic and molecular mechanisms of biofilm formation and maintenance may provide novel treatment strategies for the control of chronic infections and problems related to biofilm. Utilizing our recently developed single bacterium transcriptomic analysis technology, a Pseudomonas aeruginosa spatial gene expression profile in three distinct locations in the biofilm architecture was obtained (i.e., surface, middle, and interior of the biofilm structure). A previously uncharacterized hypothetical protein PA3966, spatially up-regulated in the interior of the biofilm, is shown to be essential for biofilm formation and in vivo pathogenesis in both mouse lung and fruit fly infection models. Additionally, PA3966 was up-regulated during chronic lung infections of cystic fibrosis patients, highlighting the significance of this protein. Further characterizations of PA3966 suggested that it is a potential attachment protein and specifically binds to two human cell lines, lung epithelial A549 cells and embryonic kidney HEK293T cells. In this study, we are proposing one aim to characterize the molecular mechanisms of this novel attachment protein. Aim 1A will identify the spatial and temporal expression patterns of PA3966 and its essential region(s) for attachment function. Aim 1B aims to validate the attachment specificity of PA3966 to host cells and identify its host cell receptor(s). Detailed characterizations of this recently discovered attachment protein PA3966 could provide potential drug-target and alternative therapeutic strategies toward better treatment outcome of bacterial biofilm related infections.