Title of Oral/Poster Presentation

Characterization of alternative purification methods of Invasion Plasmid Antigen C

Presenter Information

Abram BernardFollow

Class

Article

Department

Chemistry and Biochemistry

Faculty Mentor

Nicholas Dickenson

Presentation Type

Poster Presentation

Abstract

Shigella flexneri is a Gram-negative bacterial pathogen that presents a significant world health concern. The ability of S. flexneri to interact with and invade host membranes is crucial to its pathogenicity. Specifically, these pathogens use a type III secretion system to form a direct conduit between bacterial and host cytoplasm. This conduit allows for bacterial effectors to be secreted and elicit host responses to envelope and internalize the bacterium. Several proteins, including invasion plasmid antigen C, IpaC, localize to the tip of the T3SS needle apparatus where they are believed to play a critical role in the bacterial interaction with the host cell membrane, though direct observation and the mechanistic details of these events remain elusive. Much of this bottleneck is due to the challenge obtaining high levels of purified recombinant IpaC. Here, we present detergent-based methods used in the purification of recombinant IpaC by means of removing its co-expressed chaperone IpgC. Furthermore, we have used biophysical techniques including circular dichroism, dynamic light scattering, chemical crosslinking and size exclusion chromatography to characterize IpaC under each of the implemented conditions, finding the zwitterionic detergent LDAO appears to provide added stability and solubility characteristics to the protein compared to other methods. Finally, we tested the effect of each purification method on the ability of IpaC to both interact with and disrupt phospholipid bilayers of extruded liposomes, finding that again the LDAO purified IpaC maintained membrane "activity". Together these results identify an alternative purification method for the Shigella protein IpaC, providing a platform for additional in vitro investigation into the mechanism by which S. flexneri invades host cells.

Start Date

4-9-2015 1:30 PM

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Apr 9th, 1:30 PM

Characterization of alternative purification methods of Invasion Plasmid Antigen C

Shigella flexneri is a Gram-negative bacterial pathogen that presents a significant world health concern. The ability of S. flexneri to interact with and invade host membranes is crucial to its pathogenicity. Specifically, these pathogens use a type III secretion system to form a direct conduit between bacterial and host cytoplasm. This conduit allows for bacterial effectors to be secreted and elicit host responses to envelope and internalize the bacterium. Several proteins, including invasion plasmid antigen C, IpaC, localize to the tip of the T3SS needle apparatus where they are believed to play a critical role in the bacterial interaction with the host cell membrane, though direct observation and the mechanistic details of these events remain elusive. Much of this bottleneck is due to the challenge obtaining high levels of purified recombinant IpaC. Here, we present detergent-based methods used in the purification of recombinant IpaC by means of removing its co-expressed chaperone IpgC. Furthermore, we have used biophysical techniques including circular dichroism, dynamic light scattering, chemical crosslinking and size exclusion chromatography to characterize IpaC under each of the implemented conditions, finding the zwitterionic detergent LDAO appears to provide added stability and solubility characteristics to the protein compared to other methods. Finally, we tested the effect of each purification method on the ability of IpaC to both interact with and disrupt phospholipid bilayers of extruded liposomes, finding that again the LDAO purified IpaC maintained membrane "activity". Together these results identify an alternative purification method for the Shigella protein IpaC, providing a platform for additional in vitro investigation into the mechanism by which S. flexneri invades host cells.