Multi-drug resistant (MDR) bacterial infections represent one of the most serious challenges facing health care today. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a leading cause of hospital-acquired infections, and is particularly problematic for patients who are immunosuppressed or require mechanical ventilation. P. aeruginosa persists chronically in cystic fibrosis patients, resulting in irreversible lung damage and mortality. Both natural and acquired characteristics, including the expression of several multidrug efflux systems, make P. aeruginosa infections especially difficult to treat. In addition to its intrinsic drug resistance, the infectivity of P. aeruginosa is enhanced by expression of a Type III secretion system (T3SS). This needle-type apparatus directly injects effector proteins into eukaryotic host cells to facilitate bacterial surival and dissemination. The most cytotoxic T3SS effector produced by P. aeruginosa is the phospholipase, ExoU. This application builds upon our previous discovery that ubiquitin and ubiquitinated proteins act as essential cofactors for the activation of ExoU. The interaction of ExoU with ubiquitin is novel in that it does not involve the covalent attachment or removal of ubiquitin subunits, leading us to hypothesize that ubiquitin acts as a scaffold to induce the folding of ExoU into a catalytically-active conformation. We will employ innovative site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) methods in conjunction with genetic and biochemical approaches to characterize the structure of ExoU in solution, elucidate the structure of the ExoU-ubiquitin complex, and investigate ExoU-membrane interactions. These studies will advance the application of new methods to investigate protein-protein interactions, facilitate the development of novel inhibitors of ExoU virulence, and promote our understanding of bacterial toxins that target the membrane interface.

Public Health Relevance

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a leading cause of hospital- acquired infections, and is particularly problematic for patients who are immunosuppressed or require mechanical ventilation. P. aeruginosa persists chronically in cystic fibrosis patients, resulting in irreversible lung damage and mortaliy. The infectivity of P. aeruginosa is significantly enhanced by the Type III secreted toxin, ExoU. Our biochemical and biophysical studies of ExoU are designed to understand its molecular mechanism of activation, facilitating the development of novel inhibitors to reduce tissue damage or sepsis due to P. aeruginosa infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM114234-04
Application #
9477055
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Preusch, Peter
Project Start
2015-06-01
Project End
2019-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Biophysics
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
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
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