Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that is intrinsically resistant to antimicrobials and difficult to treat. The organism possesses a type III secretion system that is critical for evading innate immunity and establishing acute infections in compromised patients. The most toxic and destructive type III effector produced by P. aeruginosa is ExoU, whose expression is correlated with poor clinical outcomes in human infections. ExoU possesses phospholipase A2 activity, which is detectable from recombinant enzyme preparations only when a eukaryotic cofactor is provided with membrane substrates. In this new application the dynamic features of ExoU will be analyzed as a model system for understanding how the secreted T3S effectors interact with eukaryotic cofactors to affect cellular physiology. A major advance has been achieved with the discovery of ubiquitin as the main cofactor for ExoU-mediated phospholipase activity. Along with the eukaryotic cytoskeleton, the ubiquitin system is a significant target that pathogens manipulate to facilitate bacterial replication and transmission to new hosts. Ubiquitin mediated activation of ExoU represents a novel mechanism of manipulation of the host. This study is designed to functionally define the domains of ExoU involved in recognition of membrane substrates and ubiquitin. Regional and global changes in both ExoU and ubiquitin in response to cognate binding partners and liposomes will be measured utilizing state-of-the art biophysical approaches for characterizing native protein structure in solution. The completion of these aims will provide essential information towards the design of novel inhibitors and contribute biological tools to study essential eukaryotic processes and other virulence factors that are homologous to ExoU.

Public Health Relevance

During co-evolution, pathogens have exploited the essential eukaryotic ubiquitin pathways to facilitate replication and spread to the next host. Our biochemical studies of ExoU, a type III secreted phospholipase, indicate that ubiquitin is an essential cofactor for the activation of ExoU cytotoxicity. We will use the interaction of ExoU and ubiquitin to define the activation mechanism, critical structural components controlling activation and amino acid residues important for ExoU-ubiquitin interaction. This research is poised to reveal additional mechanisms for the manipulation of the host ubiquitin system, uncover new insights into mammalian cellular biology and provide unique targets and biological tools for translational applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
4R01AI104922-04
Application #
9044726
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Lu, Kristina
Project Start
2013-05-23
Project End
2017-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
4
Fiscal Year
2016
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; Sato, Hiromi; Dirck, Aaron T et al. (2015) Ubiquitin activates patatin-like phospholipases from multiple bacterial species. J Bacteriol 197:529-41
Morrow, K A; Seifert, R; Kaever, V et al. (2015) Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection. Am J Physiol Lung Cell Mol Physiol 309:L1199-207

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