Rgg proteins directly regulate gene expression and important growth phenotypes in human commensal and pathogenic bacteria, e.g., Streptococcus, Listeria, Lactocbacillus, and Enterococcus species. While it is known that Rgg proteins are DNA-binding transcriptional activators or repressors, and it is understood that peptide pheromones activate or inhibit Rgg functions that control bacterial virulence, what we are just beginning to understand is how peptide pheromones regulate the activity of Rgg receptors. However, based on our emerging understanding of pheromone activity, we have developed and fully validated productive genetic, biochemical, computational, and X-ray crystallographic approaches to identify Rgg inhibitors and determine how they function at the atomic level. The objectives in this proposal are to fully implement our screens to identify the best Rgg inhibitors for subsequent anti-infective drug development, to determine at the atomic level how Rgg function is regulated by the inhibitors as well as cognate peptide pheromones, and to explain how the conserved intermolecular disulphide bond that we discovered buried at the Rgg DNA-binding domain dimerization interface regulates pheromone receptor activity. The foundation for the proposed work is published and unpublished studies of Rgg proteins from S. pyogenes as well as other Streptococcus species performed in the applicants' laboratories. We will accomplish the objectives of this proposal by simultaneously pursuing the following two specific aims: 1) Understand at the atomic level how peptide pheromones interact with Rgg proteins and how Rgg proteins interact with DNA, determine the molecular basis of receptor activation, and reveal the role of the Rgg intermolecular disulphide bond; and 2) identify compounds that specifically inhibit Rgg protein function, determine the mechanistic basis of inhibitor activity, and test the fitness of Rgg2/3-inhibitor-resistant clones. In the first aim, a combination of X-ray crystallographic, genetic, biochemical, and computational approaches will show how the intermolecular disulphide bond and peptide pheromones regulate Rgg family proteins. The biochemical and genetic assays have been validated and an extensive number of diffracting crystals and/or preliminary structures are in hand. In the second aim, novel and highly productive high-throughput screening assays will be used to identify inhibitors of Rgg receptor function. X-ray crystallographic, genetic, biochemical, and computational approaches are in place and will be used to determine the mechanistic basis of inhibitor function and to identify the structural features of the inhibitors important for their activity. The proposed experiments are innovative because: 1) it will be shown for the first time how peptide pheromones bind and regulate Rgg receptors, 2) Rgg inhibitors will be discovered and the mechanistic basis of their activity will be described in atomic-level detail, and 3) the role of a disulphide bond in regulating a family of pheromone receptors will be elucidated. This work is significant because it will advance the field of bacterial cell-cell signaling and the development of anti-infectives.
Secreted molecules called pheromones mediate cell-cell communication between Gram-positive bacteria, which include numerous human pathogens as well as species comprising normal human microflora. Cell-cell communication commonly regulates fundamental aspects of bacterial growth, such as virulence factor expression, motility, biofilm development, antibiotic production, and genetic competence. In order to improve public health, our goals are to understand at the atomic level how Gram-positive bacteria communicate using secreted pheromones, and to develop anti-infective compounds that function to disrupt bacterial cell-cell communication.
