(Bourne Project) Toxin-Antitoxin (TA) modules are conserved interacting proteins that affect the survival of bacterial cells either by promoting the type of growth found in chronic infections, or by inducing bacterial cell death. Difficult to treat and chronic infections are increasingly prevalent, and the Centers for Disease Control estimate at least 2 million antibiotic-resistant infections occur in the United States each year at a cost of up to $20-billion in added healthcare expenses. TA modules enhance E. coli survival during antibiotic treatments, called antibiotic tolerance, which could reduce the efficacy of antimicrobial treatments. The current proposal focuses on TA modules in Pseudomonas aeruginosa, an opportunistic human pathogen of serious concern due to the prevalence of chronic and antibiotic resistant infections. Despite high variability between clinical isolates, this bacteria contains two highly conserved TA modules, 1) a RelBE/ParDE gyrase-inhibiting family module, and 2) a HigBA ribosome-inhibiting module recently shown to affect virulence production in this organism. One-third of clinical P. aeruginosa isolates contain an additional conserved ParDE system. TA modules are typically expressed as an interacting pair, thus rendering them inactive. Extracellular signals can trigger degradation of the antitoxin, releasing the toxin within the cytosol to interact with cell machinery and leading to alteration of the rate of cell replication or translation. Currently, there are fundamental gaps in our knowledge of TA module functions that prevent translation into clinical settings, including the regulation of their activation and the effect of sustained interaction of toxins with their targets. The current proposal will make significant vertical advances in the field of bacterial TA biology by directly addressing these gaps. The overall objective of the current proposal is to determine the interaction of toxins found in P. aeruginosa with their cellular targets, particularly those inhibiting DNA gyrase, and to determine the molecular interactions of the antitoxins that result in activation of these type II TA modules. The central hypothesis is that the same toxin molecule can instigate both cellular dormancy and cell death as depending on its individual potency and the length of interaction with the cellular target, and that degradation of the antitoxin is mediated by different cellular proteases depending on the specific environmental trigger.
Aim 1 will pursue the structure of the toxins interacting with DNA gyrase and will measure their potency using in vitro assays.
Aim 2 will determine the cellular proteins that interact with the antitoxins to mediate its degradation using a genetic interaction screening platform.
Aim 3 will establish the mRNA and protein levels of the three TA modules under different environmental conditions. Successful completion of these aims will determine critical steps in TA biology. In addition, mapping the cellular proteases that control the specific responses will be key in understanding the role of TA modules in bacterial physiology. These studies will directly impact the field by defining fundamental regulatory mechanisms in P. aeruginosa that contribute to antibiotic tolerance and chronic infections, thereby revealing new or synergistic antibacterial targets.

Public Health Relevance

Fundamental adaptation mechanisms of bacterial cells lead to antibacterial treatment failures due to tolerance, rather than from genetic resistance of the bacteria. Toxin-antitoxin (TA) modules are known mediators of adaptation leading to tolerance in E. coli. The current proposal will establish the molecular interactions and TA expression levels in P. aeruginosa, a prevalent opportunistic human pathogen, demonstrating the role of TA modules in adaptation responses in this bacteria and allowing advances in therapeutic developments targeting TA modules.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory Grants (P20)
Project #
Application #
Study Section
Special Emphasis Panel (ZGM1)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Oklahoma Norman
United States
Zip Code
Hebdon, Skyler D; Menon, Smita K; Richter-Addo, George B et al. (2018) Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen. J Bacteriol 200:
Cruz-Reyes, Jorge; Mooers, Blaine H M; Doharey, Pawan K et al. (2018) Dynamic RNA holo-editosomes with subcomplex variants: Insights into the control of trypanosome editing. Wiley Interdiscip Rev RNA 9:e1502
Booe, Jason M; Warner, Margaret L; Roehrkasse, Amanda M et al. (2018) Probing the Mechanism of Receptor Activity-Modifying Protein Modulation of GPCR Ligand Selectivity through Rational Design of Potent Adrenomedullin and Calcitonin Gene-Related Peptide Antagonists. Mol Pharmacol 93:355-367
Muthuramalingam, Meenakumari; White, John C; Murphy, Tamiko et al. (2018) The toxin from a ParDE toxin-antitoxin system found in Pseudomonas aeruginosa offers protection to cells challenged with anti-gyrase antibiotics. Mol Microbiol :
Roehrkasse, Amanda M; Booe, Jason M; Lee, Sang-Min et al. (2018) Structure-function analyses reveal a triple ?-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design. J Biol Chem 293:15840-15854
Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D et al. (2017) Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling. Cell Biochem Biophys 75:203-210
Van Orden, Mason J; Klein, Peter; Babu, Kesavan et al. (2017) Conserved DNA motifs in the type II-A CRISPR leader region. PeerJ 5:e3161
Murugan, Karthik; Babu, Kesavan; Sundaresan, Ramya et al. (2017) The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit. Mol Cell 68:15-25
Li, Yangxiong; Lavey, Nathan P; Coker, Jesse A et al. (2017) Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides. ACS Med Chem Lett 8:1171-1176
Wang, Bing; Powell, Samantha M; Guan, Ye et al. (2017) Nitrosoamphetamine binding to myoglobin and hemoglobin: Crystal structure of the H64A myoglobin-nitrosoamphetamine adduct. Nitric Oxide 67:26-29

Showing the most recent 10 out of 47 publications