The Pseudomonas aeruginosa type III secretion system (T3SS) functions like a molecular syringe to inject toxins into host cells. The primary cellular targets are phagocytes and epithelial cells. The injected toxins promote phagocytic avoidance and tissue destruction resulting in systemic spread of the bacteria and overwhelming sepsis. Deployment of the T3SS is highly regulated and only activated when bacteria contact host cell surfaces. The overriding hypothesis guiding the studies is that increased understanding of mechanisms that regulate T3SS gene expression will lead to the identification of critical steps that can be targeted by anti-virulence therapeutics, the long term goal of the proposed work. The primary protein required for contact- dependent activation of the T3SS is a transcription factor called ExsA. Preliminary data demonstrate that exsA transcription, ExsA synthesis, and ExsA activity are each subject to control by global regulatory systems. The goals of this proposal are to define novel mechanisms that control exsA transcription, and possibly ExsA synthesis or activity. Our preliminary data demonstrate that the 297 bp intergenic region located between exsB and exsA functions in two capacities. First, the intergenic region contains a Vfr-dependent promoter dedicated to exsA transcription. Vfr is cAMP-dependent transcription factor previously known to regulate T3SS gene expression but the mechanism has not been defined. Second, expression of the intergenic region in trans has a stimulatory effect on T3SS gene expression. Preliminary data suggest that two small peptides encoded within the intergenic region modulate T3SS gene expression. The three aims will characterize the stimulatory activity of the intergeneic region and factors that regulate intergenic promoter activity as follows:
Aim 1. Characterize the trans-acting functions of ExsF and ExsG.
Aim 2. Characterize IR promoter activity.
Aim 3. Determine whether Vfr, MvaT/MvaU, and VqsM function together, antagonistically, or independently to modulate IR promoter activity.

Public Health Relevance

Pseudomonas aeruginosa is a Gram-negative pathogen that causes life-threatening infections in humans and is reliant on a secretion system that inject toxins into host cells. The goal of the proposed studies is to dissect regulatory mechanisms that control the system, with the expectation that some of those mechanisms will prove effective targets for inhibitory therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI055042-12
Application #
9413980
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ernst, Nancy L
Project Start
2003-05-15
Project End
2021-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
12
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Iowa
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52246
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Marsden, Anne E; Intile, Peter J; Schulmeyer, Kayley H et al. (2016) Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System. J Bacteriol 198:1442-50
Marsden, Anne E; Schubot, Florian D; Yahr, Timothy L (2014) Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters. J Bacteriol 196:3546-55
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Bernhards, Robert C; Marsden, Anne E; Esher, Shannon K et al. (2013) Self-trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro. FEBS J 280:1084-94
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Brutinel, Evan D; King, Jessica M; Marsden, Anne E et al. (2012) The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties. J Bacteriol 194:2564-72

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