Pseudomonas aeruginosa is an opportunist pathogen capable of infecting a number of tissues in the human body. Its ability to cause a wide-range of infections and its resistance to antimicrobials has made P. aeruginosa one of the most threatening pathogens facing the world today. Notably, P. aeruginosa produces numerous virulence factors that lead to extreme tissue injury, and it is the long-term goal of the proposed project to combat the production of these virulence factors by antagonizing genome-wide transcription from genes regulated by the sigma factor RpoN and transcription factors known as enhancer-binding proteins (EBPs). RpoN and EBPs interact with one another to activate transcription of target genes in response to various signals. In some instances, histidine kinases sense the signals and relay that information to EBPs via phosphorylation. The overall objective of the proposed project is to characterize the EBPs and their partner histidine kinases in P. aeruginosa. Knowledge of this regulation, including the signals, target genes and regulatory mechanisms is essential to achieve the long-term goal of the proposed project. The central hypothesis is that EBPs and their partner HKs regulate an assortment of functions crucial to P. aeruginosa pathogenesis, including the utilization of various host-derived nutrients, protein secretion, production of virulence factors and alginate biosynthesis. The rationale for the proposed project is that complete knowledge of EBP regulation, i.e., the signals and target genes of EBP regulation, will provide the information needed to develop a strategy to counter P. aeruginosa pathogenesis via specifically attacking RpoN-EBP regulation.
Two specific aims are proposed to test the central hypothesis.
In Specific Aim 1, EBPs that have not yet been biochemically characterized (AauR, MifR, DdaR and EatR) or have unknown functions (PA1663 and PA1945) will be investigated. The DNA-binding specificities will be measured for each EBP, and in the case of PA1663 and PA1945, transcriptome studies will be performed to identify their potential target genes and biological functions.
In Specific Aim 2, the substrate specificities and kinetics will be measured for EBP-related histidine kinases with poorly defined mechanisms, including AauS, CbrA, DctB, KinB and MIfS. Signals or cues that stimulate the activities of these histidine kinases (and thus EBP regulation) will be identified. The approach is innovative because it will establish a complete, working model for RpoN-EBP regulation in P. aeruginosa, and importantly, define how this regulation is connected to bacterial pathogenesis on a molecular level. The proposed project is significant, because knowledge of this regulation will enable the development of antibacterial agents or compounds that can be used to combat the pathogenesis of P. aeruginosa.

Public Health Relevance

The proposed project is relevant to public health because understanding gene regulation that contributes to the virulence or pathogenesis of Pseudomonas aeruginosa will provide new inroads to combat this pathogen. Therefore, the proposed project is relevant to the part of NIH's mission that pertains to developing fundamental knowledge about the nature and behavior of living systems in order to enhance health, lengthen life, and reduce the burdens of illness.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15GM104880-03
Application #
9590849
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Ainsztein, Alexandra M
Project Start
2013-09-01
Project End
2020-06-30
Budget Start
2018-07-01
Budget End
2020-06-30
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
College of Environmental Sci & Forestry
Department
Chemistry
Type
Earth Sciences/Resources
DUNS #
152606125
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Lloyd, Megan G; Lundgren, Benjamin R; Hall, Clayton W et al. (2017) Targeting the alternative sigma factor RpoN to combat virulence in Pseudomonas aeruginosa. Sci Rep 7:12615
Lundgren, Benjamin R; Bailey, Frank J; Moley, Gabriella et al. (2017) DdaR (PA1196) Regulates Expression of Dimethylarginine Dimethylaminohydrolase for the Metabolism of Methylarginines in Pseudomonas aeruginosa PAO1. J Bacteriol 199:
Lundgren, Benjamin R; Sarwar, Zaara; Pinto, Atahualpa et al. (2016) Ethanolamine Catabolism in Pseudomonas aeruginosa PAO1 Is Regulated by the Enhancer-Binding Protein EatR (PA4021) and the Alternative Sigma Factor RpoN. J Bacteriol 198:2318-29
Sarwar, Zaara; Lundgren, Benjamin R; Grassa, Michael T et al. (2016) GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1. mSphere 1:
Sarwar, Zaara; Lundgren, Benjamin R; Grassa, Michael T et al. (2016) Erratum for Sarwar et al., GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1. mSphere 1:
Lundgren, Benjamin R; Harris, Joshua R; Sarwar, Zaara et al. (2015) The metabolism of (R)-3-hydroxybutyrate is regulated by the enhancer-binding protein PA2005 and the alternative sigma factor RpoN in Pseudomonas aeruginosa PAO1. Microbiology 161:2232-42
Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha et al. (2015) Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000. PLoS One 10:e0144852
Lundgren, Benjamin R; Villegas-Peñaranda, Luis Roberto; Harris, Joshua R et al. (2014) Genetic analysis of the assimilation of C5-dicarboxylic acids in Pseudomonas aeruginosa PAO1. J Bacteriol 196:2543-51
Lundgren, Benjamin R; Thornton, William; Dornan, Mark H et al. (2013) Gene PA2449 is essential for glycine metabolism and pyocyanin biosynthesis in Pseudomonas aeruginosa PAO1. J Bacteriol 195:2087-100