Molecular Interactions of Type III Secretion System Transcriptional Regulators in Pseudomonas aeruginosa: ExsA and ExsD

The opportunistic pathogen Pseudomonas aeruginosa ranks among the leading causes of nosocomial infections. The type III secretion system (T3SS) aids acute P. aeruginosa infections by injecting potent cytotoxins (effectors) into host cells to suppress the host's innate immune response. Expression of all T3SS-related genes is strictly dependent upon the transcription factor ExsA. Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest. The ExsA-ExsC-ExsD-ExsE signaling cascade ties host cell contact to the up-regulation of T3SS gene expression. Prior to T3SS induction, the antiactivator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC, resulting in the release of ExsA and an up-regulation of the T3SS. ExsA is an AraC/XylS-type transcriptional regulator and belongs to a subfamily of activators that regulate the T3SS in a variety of Gram-negative pathogens. These regulators are characteristically difficult to purify due to the low solubility of their C-terminal DNA binding domains. A new method for purifying ExsA was developed and produced ExsA with improved solubility. The interaction of ExsA and its PexsD promoter was examined using fluorescence anisotropy. An in vitro transcription assay was developed and it was determined that ExsA is sufficient to activate T3SS transcription. Next, the ExsD--ExsA inhibitory mechanism was examined. It was demonstrated for the first time that ExsD alone is sufficient to inhibit ExsA-dependent transcription in vitro without the aid of any other cellular factors. More significantly and contrary to previously published results, it was discovered that independently folded ExsD and ExsA are capable of interacting, but only at 37 degrees C and not at 30 degrees C. Guided by the crystal structure of ExsD, a monomeric variant of the protein was designed to demonstrate that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30 degrees C. To further elucidate the ExsD-ExsA inhibitory mechanism, the ExsD-ExsA interface was examined. ExsD variants were generated and used to determine which region of ExsD interacts with ExsA. Interestingly, ExsD was also found to bind DNA, although it is unclear whether or not this plays a role in ExsA inhibition. Fully understanding the mechanism by which ExsD inhibits ExsA may enable the development of drugs that target ExsA in order to shut down the T3SS, thereby eliminating P. aeruginosa infection.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biological Sciences

Department dc:contributor.department

Biological Sciences

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2013

Author dc:creator

• Bernhards, Robert Cory

Chair dc:contributor.committeechair

• Schubot, Florian D.

Committee members dc:contributor.committeemember

• Stevens, Ann M.
• Melville, Stephen B.
• Hernick, Marcy

• Pseudomonas aeruginosa
• ExsA
• ExsD
• type III secretion system
• transcriptional regulation

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

vt_gsexam:664

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/50928