Cyclic di-GMP (c-di-GMP) is a near-ubiquitous, newly appreciated second messenger signal in bacteria that contributes to pathogenicity-promoting behaviors including biofilm formation, motility, virulence factor expression, development, and quorum sensing. Its signaling pathways are thus potentially attractive targets for new approaches to combat biofilm-based or acute infections, but the mechanisms by which it regulates transcription in bacteria are largely unknown. The goal of our research is to elucidate, and thus potentially enable therapeutic targeting of, the mechanisms that mediate c-di-GMP signaling in bacteria by integrating genetic, biochemical, chemical, structural, bioinformatic, and computational approaches. We and others have previously found that a subset of transcription factors belonging to the NtrC-like bacterial enhancer binding protein (EBP) family directly bind and respond to c-di-GMP. EBPs are widespread in bacteria, and regulate fundamental bacterial behaviors including biofilm formation, motility, quorum sensing, and virulence factor expression. We further found that c-di-GMP binds to and inhibits the ability of the Vibrio cholerae ?54- dependent EBP FlrA to induce motility. Our preliminary data suggest that c-di-GMP inhibits transcription by locking dimeric FlrA into a conformation incapable of DNA binding, but conversely binds to and activates the ?70-dependent V. cholerae EBP VpsR to induce biofilm formation. We hypothesize that c-di-GMP activates transcription by stimulating VpsR oligomerization.
In Aims 1 & 2 we will test these hypotheses, using combined in vivo and in vitro genetic and biochemical assays to identify critical structural determinants for this regulation and define the impact of c-di-GMP on transcription factor activity. These studies will be integrated with the elucidation of the X-ray crystal structures of FlrA and VpsR in the presence and absence of c-di-GMP binding to formulate a mechanistic model of c-di-GMP regulation of EBPs. Elucidating these mechanisms will allow us to identify among the thousands of EBPs in diverse bacterial species those that are c-di-GMP-regulated. Preliminary studies generating crystals of purified FlrA proteins, and the identification of c-di-GMP-insensitive, constitutively active FlrA and VpsR mutants support the feasibility of these studies.
In Aim 3 we will expand our analysis to identify novel c-di-GMP-dependent transcriptional machinery in V. cholerae and completely define the c-di-GMP-dependent regulatory network. This analysis will fully harness newly developed deep sequencing technologies (TN-seq, RNA-seq, and IPODHR). We will use these data to formulate a computation model of the c-di-GMP regulon in V. cholerae, gaining an appreciation for the global impact of c-di-GMP on this pathogen and uncovering fundamental principles that generally underpin c-di-GMP regulatory networks. Our studies will advance current concepts of the control of bacterial transcriptional initiation, identifying novel targets or development of new antibiotics that are agonists or antagonists of c-di-GMP-mediated regulation in pathogenic bacterial species.

Public Health Relevance

The bacterial second messenger cyclic di-GMP is a key regulator of biofilm formation and virulence factor expression in pathogenic bacteria. Here, we will elucidate fundamental mechanisms by which c-di-GMP regulates transcription in the human pathogen Vibrio cholerae, which will provide new targets for therapeutic intervention of cyclic di-GMP signaling to prevent bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM109259-01A1
Application #
8887427
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
2015-07-01
Project End
2020-06-30
Budget Start
2015-07-01
Budget End
2016-06-30
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Michigan State University
Department
Microbiology/Immun/Virology
Type
Schools of Osteopathic Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Waters, Christopher M (2018) Shining the Light on Cyclic di-GMP Dark Matter. J Bacteriol 200:
Agostoni, Marco; Logan-Jackson, Alshaé R; Heinz, Emily R et al. (2018) Homeostasis of Second Messenger Cyclic-di-AMP Is Critical for Cyanobacterial Fitness and Acclimation to Abiotic Stress. Front Microbiol 9:1121
Fernandez, Nicolas; Waters, Christopher M (2018) Analyzing Diguanylate Cyclase Activity In Vivo using a Heterologous Escherichia coli Host. Curr Protoc Microbiol :e74
Maiden, Michael M; Hunt, Alessandra M Agostinho; Zachos, Mitchell P et al. (2018) Triclosan Is an Aminoglycoside Adjuvant for Eradication of Pseudomonas aeruginosa Biofilms. Antimicrob Agents Chemother 62:
Severin, Geoffrey B; Ramliden, Miriam S; Hawver, Lisa A et al. (2018) Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae. Proc Natl Acad Sci U S A 115:E6048-E6055
Fernandez, Nicolas L; Srivastava, Disha; Ngouajio, Amanda L et al. (2018) Cyclic di-GMP Positively Regulates DNA Repair in Vibrio cholerae. J Bacteriol 200:
Berne, Cécile; Ellison, Courtney K; Agarwal, Radhika et al. (2018) Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA. Mol Microbiol 110:219-238
Pursley, Benjamin R; Maiden, Michael M; Hsieh, Meng-Lun et al. (2018) Cyclic di-GMP Regulates TfoY in Vibrio cholerae To Control Motility by both Transcriptional and Posttranscriptional Mechanisms. J Bacteriol 200:
Bruger, Eric L; Waters, Christopher M (2018) Maximizing Growth Yield and Dispersal via Quorum Sensing Promotes Cooperation in Vibrio Bacteria. Appl Environ Microbiol 84:
Yuan, Xiaochen; Tian, Fang; He, Chenyang et al. (2018) The diguanylate cyclase GcpA inhibits the production of pectate lyases via the H-NS protein and RsmB regulatory RNA in Dickeya dadantii. Mol Plant Pathol :

Showing the most recent 10 out of 20 publications