The molecule cyclic dimeric GMP (c-di-GMP) has emerged as a broadly conserved second messenger in bacteria, controlling adhesion, motility, biofilm formation and cell morphogenesis in diverse bacterial species, while exerting control at transcriptional, translational and post-translational levels. A key, recent advance in our understanding of this nucleotide's role in bacteria has been the identification of c-di-GMP receptors with defined outputs. Our studies have established a model wherein control of adhesion protein localization on the bacterial cell surface is mediated by cytoplasmic levels of c-di-GMP. c-di-GMP levels are monitored by LapD, an inner membrane-localized c-di-GMP effector. LapD switches between a c-di- GMP-bound on-state and a nucleotide-free off-state. Depletion of cellular c-di-GMP results in the dissociatio of c-di-GMP from LapD, which in turn propagates a signal across the inner membrane and to the periplasmic domain of LapD. At high cytosolic c-di-GMP levels, LapD sequesters the periplasmic protease LapG, preventing it from processing its substrate, a large adhesion protein LapA at the cell surface, and from releasing it from the cell surface, and thus promoting biofilm formation. The proposed research builds upon ongoing collaborative studies in the O'Toole and Sondermann labs to explore the conservation and mechanistic basis whereby the LapD/LapG signal transduction system regulates the localization of bacterial surface proteins.
In Aim 1, we will test the hypothesis that c-di-GMP binding to the cytoplasmic domain of LapD causes structural rearrangements of this effector in a wide range of bacteria including important pathogens.
In Aim 2, we will test the hypothesis that LapD-mediated control of LapG is dependent on a conserved, direct protein-protein interaction. This interaction is a pivotal point for the development for pharmacological tools to interfere with biofilm formation or virulence.
Aim 3 will test the hypothesis that LapG recognizes discrete features of the N-terminal domain of LapA that allows this protease to specifically target the adhesin. Core principles established by this work will be applicable to a wide range of bacterial cell surface receptors. In addition, elucidating the regulatory principles that control bacterial cell adhesion via the LapDGA system will be invaluable for targeting the underlying molecular interactions and mechanisms pharmacologically. While the research described here is basic in nature, it will have ramifications for infection biology research pertaining to pathogens and their associated diseases such as Pseudomonas aeruginosa (cystic fibrosis, hospital-acquired infections), Vibrio cholerae (cholera) and Legionella pneumophila (Legionnaires' disease). Ultimately, we hope that our efforts will help to counteract the increasing disparity between the emergence of drug-resistant bacteria and the decline in novel therapeutics.

Public Health Relevance

The bacterial second messenger c-di-GMP plays a critical role in the control of the formation of bacterial communities known as biofilms. These biofilms have a profound negative impact in clinical settings, causing chronic infections that extend hospital stays and raise healthcare costs We have identified a regulatory system that controls the function of a key adhesion required to make biofilms in a wide range of microbes. In this proposal, we will investigate critical molecular mechanisms of this signaling system, which may contribute to new anti-biofilm and anti-infective therapies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Huntley, Clayton C
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Cornell University
Other Basic Sciences
Schools of Veterinary Medicine
United States
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Dahlstrom, Kurt M; O'Toole, George A (2017) A Symphony of Cyclases: Specificity in Diguanylate Cyclase Signaling. Annu Rev Microbiol 71:179-195
Krasteva, Petya Violinova; Sondermann, Holger (2017) Versatile modes of cellular regulation via cyclic dinucleotides. Nat Chem Biol 13:350-359
Cooley, Richard B; Sondermann, Holger (2017) Probing Protein-Protein Interactions with Genetically Encoded Photoactivatable Cross-Linkers. Methods Mol Biol 1657:331-345
Conner, Jenna G; Zamorano-Sánchez, David; Park, Jin Hwan et al. (2017) The ins and outs of cyclic di-GMP signaling in Vibrio cholerae. Curr Opin Microbiol 36:20-29
Cooley, Richard B; O'Donnell, John P; Sondermann, Holger (2016) Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor. Elife 5:
Heussler, Gary E; O'Toole, George A (2016) Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems. J Bacteriol 198:1481-6
Dahlstrom, Kurt M; Giglio, Krista M; Sondermann, Holger et al. (2016) The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein. J Bacteriol 198:1595-603
Cooley, Richard B; Smith, T Jarrod; Leung, Wilfred et al. (2016) Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate. J Bacteriol 198:66-76
Matsuyama, Bruno Y; Krasteva, Petya V; Baraquet, Claudine et al. (2016) Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 113:E209-18
Dahlstrom, Kurt M; Giglio, Krista M; Collins, Alan J et al. (2015) Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector. MBio 6:e01978-15

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