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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI097307-01A1
Application #
8369667
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Korpela, Jukka K
Project Start
2012-05-15
Project End
2017-04-30
Budget Start
2012-05-15
Budget End
2013-04-30
Support Year
1
Fiscal Year
2012
Total Cost
$397,549
Indirect Cost
$74,549
Name
Cornell University
Department
Other Basic Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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
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
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
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
Ha, Dae-Gon; Richman, Megan E; O'Toole, George A (2014) Deletion mutant library for investigation of functional outputs of cyclic diguanylate metabolism in Pseudomonas aeruginosa PA14. Appl Environ Microbiol 80:3384-93
El-Kirat-Chatel, Sofiane; Boyd, Chelsea D; O'Toole, George A et al. (2014) Single-molecule analysis of Pseudomonas fluorescens footprints. ACS Nano 8:1690-8
El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Boyd, Chelsea D et al. (2014) Single-cell and single-molecule analysis deciphers the localization, adhesion, and mechanics of the biofilm adhesin LapA. ACS Chem Biol 9:485-94
Boyd, Chelsea D; Smith, T Jarrod; El-Kirat-Chatel, Sofiane et al. (2014) Structural features of the Pseudomonas fluorescens biofilm adhesin LapA required for LapG-dependent cleavage, biofilm formation, and cell surface localization. J Bacteriol 196:2775-88
Chatterjee, Debashree; Cooley, Richard B; Boyd, Chelsea D et al. (2014) Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP. Elife 3:e03650

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