Persistent bacterial infections are a major cause of death in cystic fibrosis patients and immune-compromised individuals. A number of gram-negative bacteria including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis, cause infections that are difficult to treat because the bacteria form a """"""""biofilm community"""""""" that renders them less sensitive to traditional antibiotics. Quorum sensing, mediated by acylhomoserine lactone (AHL) signaling molecules, regulates pathogenesis and biofilm formation in P. aeruginosa. Therefore, understanding the molecular basis of quorum sensing is a high priority in the development of novel anti-bacterial agents. The long term goal of this project is to extend the understanding of the quorum-sensing system to the atomic level to develop a detailed description of the mechanisms that control bacterial pathogenesis. The main focus of this proposal is the class of enzymes that produce the AHL signal, AHL-synthases, because bacteria lacking the AHL signal fail to become pathogenic or form stable biofilms. Although there are models of the mechanism of action of the AHL-synthases, there are currently no structures of any AHL synthase. High resolution structural information is absolutely essential for fully understanding the mechanism of AHL synthesis and will provide the basis for future structure-based inhibitor design for development of novel therapeutics.
The specific aims for this project are: (I) determine the high resolution crystal structure of the Pantoea stewartii subsp. Stewartii AHL-synthase (EsaI) to understand its function, mechanism, and relationship to other enzymes that utilize similar substrates. Perform mutagenesis, binding and kinetics experiments with EsaI to better understand the catalytic mechanism and substrate specificity. (II) Study the P. aeruginosa AHL-synthase, LasI, using structural and biochemical techniques to understand how specificity of AHL production is determined. (III) Establish whether the AHL-synthase homologues in divergent organisms produce a homoserine lactone signal using mass spectrometry and activity assays. Study the structures and mechanisms to determine similarities to other AHL synthases.
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