Pertussis is a re-emerging public health concern, and its resurgence is correlated with a switch from whole-cell to acellular vaccines, which prevent disease, but not colonization or transmission of the causative agent Bordetella pertussis. In order to limit the mortality and morbidity associated with pertussis, particularly in infants, a combination of more effective vaccines and antibiotic therapies are needed. Their development requires a better understanding of the molecular basis for Bordetella pertussis pathogenicity. We are proposing experiments that will answer longstanding questions regarding the secretory mechanism of Filamentous hemagglutinin (FhaB), a critical virulence factor for Bordetella pertussis and to identify peptides which inhibit FhaB secretion. FhaB mediates adherence to mammalian respiratory epithelial cells, suppresses the inflammatory response and is required for Bordetella to persist in the murine respiratory tract. FhaB is a TpsA protein secreted by the TpsB protein FhaC. FhaBC are the model for two-partner secretion (TPS) systems. TPS systems are broadly distributed among Gram-negative bacteria and many have proven or postulated roles in virulence. TpsB proteins are part of the Omp85 superfamily which also includes outer membrane protein assembly factors such BamA. Due to the complex topology and large size of FhaB and other TpsA effectors, no structures of active TpsB transporters in complex with their effectors have been solved. The lack of structural information has limited our understanding of how TpsB proteins interact with TpsA passengers and complicates the design of effective anti-microbials targeting these systems. We propose to: 1) Determine the mechanism and structure of the early stages of FhaB secretion by FhaC using a simplified FhaB/FhaC system that we have developed in E. coli and 2) screen a large library (>1012) of cyclic peptides to identify those that bind and inhibit FhaC. This is a collaborative project that takes advantage of the expertise and resources of the Cotter and Doyle laboratories. The results of this work will lay the foundation for a substantial collaboration between the two groups investigating the molecular basis for the TPS mechanism and identification of antimicrobial compounds targeting these systems in Gram negative pathogens.