Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is problematic for individuals who are immunocompromised. Importantly, P. aeruginosa is responsible for pathology associated with chronic colonization of individuals with cystic fibrosis. The organism is well adapted to health care settings by being able to survive on minimal nutrients, form microbial communities on surfaces and express intrinsic resistance to antibiotics and disinfectants. P. aeruginosa has a large repertoire of destructive enzymes and toxins that aid bacterial replication by neutralizing innate immune cells in human hosts. The mechanisms of action include interference with or inhibition of signal transduction, host protein synthesis, cytoskeletal function or membrane dynamics. Our studies focus on the cytotoxic enzymes or effectors injected by the type III secretion system of P. aeruginosa. These enzymes, ExoS and ExoU, demonstrate broad substrate specificity and can recognize both prokaryotic and eukaryotic targets. The property of broad substrate specificity requires that additional control elements must be in place to keep the effector inactive before delivery to the appropriate host environment. The type III effector, ExoU, a potent phospholipase, is used as a model system to study the dynamic changes that mediate activation of enzyme activity in host cells. We identified ubiquitin (Ub), which is synthesized only by eukaryotes, as the activator required for ExoU- mediated phospholipase activity in host cells. The long-term objectives of this research are to mechanistically describe Ub and substrate induced conformational changes that occur to activate ExoU. We have used molecular modeling, continuous wave and double electron electron resonance spectroscopy to build an initial model of ExoU activation. The model makes specific predictions and provides testable hypotheses that will be challenged in iterative biochemical and biophysical analyses and model refinement with novel mono- and diUb probes. Ub-mediated activation is postulated to serve as a specific, therapeutic target, not limited to ExoU. This property extends to ExoU-orthologs that are encoded in the genomes of a variety of Gram negative human pathogens and opportunists. Overall, these studies aim to contribute a structure-function basis for the rational design of inhibitors that may be applicable for treatment of infections caused by resistant, problematic organisms.

Public Health Relevance

Pseudomonas aeruginosa and a variety of bacterial genera deliver effectors directly into host cells via a specialized injection system. Effectors encode toxic enzymatic activities that cause cell death or dysfunction leading to pathology, tissue destruction and poor outcomes for infected individuals. The long-term goals of this research are to identify how a host encoded cofactor, ubiquitin, interacts with the P. aeruginosa effector, ExoU. Mapping this interface will provide information needed to design inhibitors that may be active against a variety of bacteria, most of which are highly resistant to antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI104922-07
Application #
9762818
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Lu, Kristina
Project Start
2013-05-23
Project End
2022-08-31
Budget Start
2019-09-01
Budget End
2020-08-31
Support Year
7
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
937639060
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Feix, Jimmy B; Kohn, Samantha; Tessmer, Maxx H et al. (2018) Conformational Changes and Membrane Interaction of the Bacterial Phospholipase, ExoU: Characterization by Site-Directed Spin Labeling. Cell Biochem Biophys :
Tessmer, Maxx H; Anderson, David M; Pickrum, Adam M et al. (2018) Identification of a ubiquitin-binding interface using Rosetta and DEER. Proc Natl Acad Sci U S A 115:525-530
Balczon, Ron; Morrow, K Adam; Zhou, Chun et al. (2017) Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids. FASEB J 31:2785-2796
Tessmer, Maxx H; Anderson, David M; Buchaklian, Adam et al. (2017) Cooperative Substrate-Cofactor Interactions and Membrane Localization of the Bacterial Phospholipase A2 (PLA2) Enzyme, ExoU. J Biol Chem 292:3411-3419
Fischer, Axel W; Anderson, David M; Tessmer, Maxx H et al. (2017) Structure and Dynamics of Type III Secretion Effector Protein ExoU As determined by SDSL-EPR Spectroscopy in Conjunction with De Novo Protein Folding. ACS Omega 2:2977-2984
Morrow, K Adam; Frank, Dara W; Balczon, Ron et al. (2017) The Pseudomonas aeruginosa Exoenzyme Y: A Promiscuous Nucleotidyl Cyclase Edema Factor and Virulence Determinant. Handb Exp Pharmacol 238:67-85
Torres, Iviana M; Demirdjian, Sally; Vargas, Jennifer et al. (2017) Acidosis increases the susceptibility of respiratory epithelial cells to Pseudomonas aeruginosa-induced cytotoxicity. Am J Physiol Lung Cell Mol Physiol 313:L126-L137
Morrow, K Adam; Ochoa, Cristhiaan D; Balczon, Ron et al. (2016) Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy. Am J Physiol Lung Cell Mol Physiol 310:L337-53
Anderson, David M; Feix, Jimmy B; Frank, Dara W (2015) Cross Kingdom Activators of Five Classes of Bacterial Effectors. PLoS Pathog 11:e1004944
Anderson, David M; Sato, Hiromi; Dirck, Aaron T et al. (2015) Ubiquitin activates patatin-like phospholipases from multiple bacterial species. J Bacteriol 197:529-41

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