Confirming the Importance of C-terminal residues of Pseudomonas aeruginosa exotoxin ExoU for chaperone interaction by copurification
Advisor Information
Donald Rowen
Location
Dr. C.C. and Mabel L. Criss Library
Presentation Type
Poster
Start Date
4-3-2016 10:45 AM
End Date
4-3-2016 12:15 PM
Abstract
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for approximately 10% of hospital-acquired infections. One of the virulence factors of P. aeruginosa is the exotoxin ExoU, which is injected into the cytoplasm of target cells through a needle-like Type III secretion system. Exposure to as little as a few hundred molecules of ExoU will rapidly induce cytolytic cell death. Effective secretion of the ExoU toxin requires interactions with its cognate chaperone SpcU. Binding to the chaperone stabilizes the ExoU structure, facilitating the delivery of inactive toxin to the Type III secretion system. However, it is unclear which specific residues of the ExoU protein are necessary for proper binding of the toxin to its chaperone. Previous studies in our lab suggested that residues on both the C-terminus and the N-terminus of ExoU may be required for interaction with SpcU. To confirm the importance of the C-terminal residues, we determined whether two mutants of ExoU lacking different portions of the C-terminal of ExoU would bind to and be copurified with His-tagged SpcU. Western blots indicated that the copurificaiton of ExoU with His-SpcU was reduced by both C-terminal truncations, which supports our hypothesis that residues on the C-terminus of ExoU are required for SpcU binding.
Confirming the Importance of C-terminal residues of Pseudomonas aeruginosa exotoxin ExoU for chaperone interaction by copurification
Dr. C.C. and Mabel L. Criss Library
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for approximately 10% of hospital-acquired infections. One of the virulence factors of P. aeruginosa is the exotoxin ExoU, which is injected into the cytoplasm of target cells through a needle-like Type III secretion system. Exposure to as little as a few hundred molecules of ExoU will rapidly induce cytolytic cell death. Effective secretion of the ExoU toxin requires interactions with its cognate chaperone SpcU. Binding to the chaperone stabilizes the ExoU structure, facilitating the delivery of inactive toxin to the Type III secretion system. However, it is unclear which specific residues of the ExoU protein are necessary for proper binding of the toxin to its chaperone. Previous studies in our lab suggested that residues on both the C-terminus and the N-terminus of ExoU may be required for interaction with SpcU. To confirm the importance of the C-terminal residues, we determined whether two mutants of ExoU lacking different portions of the C-terminal of ExoU would bind to and be copurified with His-tagged SpcU. Western blots indicated that the copurificaiton of ExoU with His-SpcU was reduced by both C-terminal truncations, which supports our hypothesis that residues on the C-terminus of ExoU are required for SpcU binding.