Biofilms are surface-attached microbial communities found in clinical, industrial and natural environments. Biofilms negatively impacts human health, in particular the formation of antibiotic resistant biofilms on a broad range of medical implants such as catheters, orthopedic implants and contact lenses is a significant clinical problem. Our previous studies demonstrated that the nucleotide signal cyclic diguanylate (cdG) controls biofilm formation via control of extracellular polysaccharide (EPS) and flagellar function. In this renewal application, we analyze a signaling pathway required for the control of flagellar function during early biofilm formation by the opportunistic pathogen P. aeruginosa. Our central hypothesis is that cdG levels are up-regulated in response to cell-to-substratum contact, leading to reduced motility and promotion of biofilm formation. In particular, we propose a """"""""surface-sensing"""""""" pathway that depends on the PilY1 protein, with subsequent increase of cdG, dynamic swapping of two distinct flagellar stators triggered by increased cdG, and, finally, reduced motility, favoring biofilm formation. In this application, we use community level and single cell analyses to explore the mechanistic underpinnings of the earliest events in biofilm formation. A more complete understanding of early events in biofilm formation by this opportunistic pathogen, and a more general understanding of cdG signaling, will provide insights into how to prevent and treat infections caused by a broad range of biofilm-forming microbes.
Our Specific Aims are:
Specific Aim 1. Test the hypothesis that early biofilm formation is promoted by surface-dependent stimulation of cdG levels mediated by PilY1, a component of a putative surface-sensing pathway.
Specific Aim 2. Test the hypothesis that a newly described signal transduction pathway regulates early biofilm formation through production and binding of cdG to control flagellar-mediated surface motility.
Specific Aim 3. Test the hypothesis that dynamic stator exchange down-regulates flagellar motility on a surface in response to cdG.

Public Health Relevance

Biofilms are surface-attached microbial communities found in clinical, industrial and natural environments. The formation of biofilms negatively impacts human health, in particular the formation of antibiotic tolerant biofilms on a broad range of medical implants such as catheters and contact lenses. Device related infections cost the healthcare industry hundreds of millions of dollars annually in extended hospital stays, therapy and associated complications. Recent studies also suggest that biofilm formation plays a role in non-implant infections, for example, in addition to well-documented studies of plaque, there is emerging evidence that biofilms on host tissues in Cystic Fibrosis, otitis media (e.g., ear ache) and endocarditis. We have identified a regulatory network that regulates biofilm formation in the important opportunistic pathogen P. aeruginosa. A better understanding of this network may provide new targets for blocking the formation of these communities on medical implants and on host tissues.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (ZRG1)
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Taylor, Christopher E,
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Dartmouth College
Schools of Medicine
United States
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Lee, Calvin K; de Anda, Jaime; Baker, Amy E et al. (2018) Multigenerational memory and adaptive adhesion in early bacterial biofilm communities. Proc Natl Acad Sci U S A 115:4471-4476
O'Toole, George A (2018) Cystic Fibrosis Airway Microbiome: Overturning the Old, Opening the Way for the New. J Bacteriol 200:
Limoli, Dominique H; Whitfield, Gregory B; Kitao, Tomoe et al. (2017) Pseudomonas aeruginosa Alginate Overproduction Promotes Coexistence with Staphylococcus aureus in a Model of Cystic Fibrosis Respiratory Infection. MBio 8:
de Anda, Jaime; Lee, Ernest Y; Lee, Calvin K et al. (2017) High-Speed ""4D"" Computational Microscopy of Bacterial Surface Motility. ACS Nano 11:9340-9351
Ribbe, Jan; Baker, Amy E; Euler, Sebastian et al. (2017) Role of Cyclic Di-GMP and Exopolysaccharide in Type IV Pilus Dynamics. J Bacteriol 199:
Orazi, Giulia; O'Toole, George A (2017) Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of Cystic Fibrosis Infection. MBio 8:
Bernier, Steve P; Hum, Courtney; Li, Xiang et al. (2017) Pseudomonas aeruginosa-Derived Rhamnolipids and Other Detergents Modulate Colony Morphotype and Motility in the Burkholderia cepacia Complex. J Bacteriol 199:
Baker, Amy E; O'Toole, George A (2017) Bacteria, Rev Your Engines: Stator Dynamics Regulate Flagellar Motility. J Bacteriol 199:
Armitage, Judith P; Becker, Anke; Christie, Peter J et al. (2017) Classic Spotlights: Selected Highlights from the First 100 Years of the Journal of Bacteriology. J Bacteriol 199:
O'Toole, George A (2017) Special Meeting Sections for the ASM Conference on Mechanisms of Interbacterial Cooperation and Competition. J Bacteriol 199:

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