While over $25 billion is spent to treat biofilm-based infections in the United States annually, very little is known about the proteins in the biofilm matrix, which protects the resident bacteria from antimicrobial attack. The long-term goal of this application and the Candidate, Dr. Tseng, is to establish an independent research program centered on understanding the function of extracellular matrix-associated proteins in biofilm formation and antimicrobial tolerance. Towards this goal, the immediate career objective of Dr. Tseng is to obtain an independent faculty position, using the research proposed in this application as the cornerstone of her job applications. The overall research objective of this application is to determine the role of ecotin, a serine protease inhibitor that is a putative matrx-associated protein, in the biofilm matrix. The hypothesis is that by inhibiting proteases, ecotin i the biofilm matrix regulates biofilm formation and protects the biofilm from exogenous proteolytic attack. To test this hypothesis, two specific aims are proposed: 1) to evaluate the role of ecotin in protecting the biofilm bacteria, using a diabetic mouse wound model of chronic biofilm infections (in consultation with Dr. Singh) and an in vitro flow cell biofilm model; and 2) to examine the role of ecotin in biofilm formation via immunofluorescence to examine its matrix localization, via qRT-PCR and immunoblot to determine its biofilm expression pattern, and via comparative proteomics of biofilms to examine its function. These aims are expected to demonstrate that enzymatic activities of matrix-associated proteins are important for biofilm formation and antimicrobial tolerance. In addition, the outcomes of these aims are expected to provide preliminary data for a competitive R01 application. The rationale for this research is that it is expected to yield important new insights into biofilm biology, while also providing the means necessary to establish Dr. Tseng, who is committed to a career in basic biomedical research, as an independent molecular microbiologist. In addition to the research described above, this proposal includes career development activities to complement Dr. Tseng's prior experience, as well as an advisory committee assembled by Dr. Tseng. Dr. Parsek, the postdoctoral advisor of Dr. Tseng, is included in this application as a scientific advisor. He is ideal for this position, s he is an internationally recognized expert in biofilm biology with a strong track record of producing successful independent academic scientists. Finally, this Career Transition Award is expected to aid Dr. Tseng in the transition to independence and provide preliminary data for an R01 application within the two years of this award.