Exploration of quorum sensing in tuberculosis

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PROJECT SUMMARY Mycobacterium are responsible for many important human diseases, including tuberculosis and leprosy. Bacterial species use inter-bacterial signaling or quorum sensing (QS) to coordinate gene regulation in response to their environment. Despite this fact and the presence of numerous QS molecule responsive regulators (LuxR homologues) in all mycobacterial genomes sequenced, no quorum sensing molecules have been identified in any mycobacterial species. The only potential signaling molecule yet identified is resuscitation promoting factor (Rpf) that is a lysozyme-like protein that can stimulate reactivation of latent tuberculosis. As an exploratory project, we set out in search of the best way to identify QS molecules from mycobacteria. Most likely due to the extremely low concentrations of QS molecules required to signal, nearly all QS molecules have been identified through use of a sensor strain that produces light or pigment upon exposure to QS molecules. Since most bacterial sensor strains are Gram negatives, making it unsurprising that they have not been used successfully to identify mycobacterial QS molecules, we searched for a new sensor system. We reasoned that a sensor based on Streptomyces, an Actinomycetales closely related to Mycobacterium, would be likely to respond to mycobacterial QS molecules. In our preliminary studies, we found that Streptomyces griseus and S. coelicolor can respond to mycobacterial QS molecules. The use of a novel QS sensor strain allowed us to purify two of these molecules, designated MAI-1 and MAI-2, determine partial structure for MAI-1 and demonstrate impacts of MAI-1 and MAI-2 on virulence-related phenotypes. In this application we propose to further analyze the QS pathways in mycobacteria to determine their role in pathogenesis. This will be accomplished through two specific aims: 1) Identification of mycobacterial QS molecules. Our working hypothesis is that mycobacterial QS molecules are structurally related to γ- butyrolactone (GBL) QS molecules in Streptomyces. Our preliminary studies demonstrate that MAI-1 and MAI-2 can trigger QS pathways in S. coelicolor, suggesting that they are related to each other and to GBLs, but we have not yet fully determined their structures. In this aim, we will utilize our novel sensor to determine their structures and confirm their ability to function as signaling molecules. 2) Dissect the mechanisms of QS signaling in mycobacteria. Our working hypothesis is that MAI-1 and MAI-2 are made by mycobacteria to control gene expression in density-dependent fashion. Our preliminary studies developed a screen for production of MAI molecules by mycobacteria and found that MAI molecules enhance biofilm formation, colonization of epithelial cells and macrophages and growth in macrophages. We will analyze the pathways involved in synthesis of these QS molecules in mycobacteria. The overall goal of these studies is to understand how these QS molecules are made and affect the ability of mycobacteria to cause disease.