The role of master regulator NtrC in amyloid fibril dependent pathogenic traits of Pseudomonas aeruginosa

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Project Summary/Abstract Pseudomonas aeruginosa is a versatile pathogen, capable of infecting numerous human tissues and inflicting irreversible, life-threatening damage. Not only can P. aeruginosa survive on the nutrients released from the damaged host cells and/or tissues, but importantly, this bacterium has the propensity to adapt and protect itself from any defensive measures taken against it whether from the immune response of the host organism or external remedies such as antibiotic treatments. It is the long-term goal of the proposed project to neutralize or counter these key aspects of P. aeruginosa pathogenesis through the specific targeted disruption of the enhancer-binding protein (EBP)-RpoN regulatory network. RpoN and EBPs interact with each other to activate transcription of >200 target genes in P. aeruginosa. Many of the EBP-RpoN targeted genes are essential for the utilization of host-derived nutrients and provide resistance against cell-damaging agents, including peroxides, heat and antibiotics. The central hypothesis of the proposal is that the EBP NtrC regulates the expression of amyloid fibrils − as well as additional proteins − that allow for P. aeruginosa to bind to eukaryotic cells and gain a foothold in the host environment. The rationale for the proposed study is that knowledge of both the gene- regulatory networks and pathogenic roles for NtrC will provide crucial insight into the molecular mechanisms and signals that coordinate the adaptation of P. aeruginosa to a eukaryotic-host environment. In Specific Aim 1, the regulon of NtrC will be defined. The chromosomal DNA-binding sites for NtrC will be determined via chromatin immunoprecipitation with exonuclease treatment-high-throughput sequencing (ChIP-exo). Additionally, the transcriptome of NtrC will be identified using RNA-seq. The results of the ChIP-exo and RNA-seq together will identify the gene-regulatory network directly governed by NtrC. In Specific Aim 2, the roles of amyloid fibrils and its regulator NtrC will be investigated in terms of adhesion and cytotoxicity towards eukaryotic cells. The approach is innovative because it will establish whether amyloid fibrils and its regulator NtrC are required for cell adhesion of P. aeruginosa in a host environment. It will also serve as a template for using ChIP-exo in the characterization of EBPs or DNA-binding proteins in other pathogenic microorganisms. We are getting closer to having a complete definition of EBP-RpoN regulation in this important human pathogen. The proposed project is significant, because knowledge of this regulation and the resistance mechanisms it controls will facilitate the development of antimicrobials and alternative approaches to counter the virulence or pathogenesis of P. aeruginosa.