Unraveling the Biosynthesis of Aromatic Virulence Factors in Pseudomonas aeruginosa

/Abstract 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 towards antimicrobials has made P. aeruginosa one of the most threatening pathogens facing the world today. P. aeruginosa produces numerous virulence factors that cause extreme tissue injury, and it is the long-term goal of the proposed project to understand at the molecular level the essential enzymatic reaction that leads to the biosynthesis of all aromatic virulence factors, including phenazines and PQS (Pseudomonas quinolone signal). This essential reaction is the condensation of phosphoenolpyruvate with erythrose 4-phosphate to yield 3-deoxy-D-arabino-heptulosonate 7-phosphate or DAHP. This reaction is catalyzed by DAHP synthases and is the first committed step in the biosynthesis of all aromatic compounds in bacteria. The overall objective of the proposed project is to characterize the DAHP synthases of P. aeruginosa. The central hypothesis is that the DAHP synthases of P. aeruginosa contribute differently to the biosynthesis of aromatic virulence factors. Specifically, two DAHP synthases (PA1750 and PA2943) are proposed to be required for generating DAHP used in the biosynthesis of aromatic amino acids and vitamins, whereas another pair of DAHP synthases (PhzC1 and PhzC2) are expected to be essential for generating DAHP used to produce phenazines and PQS. The specific role(s) of each DAHP synthase is hypothesized to be a direct result of its unique catalytic properties and/or expression levels. The rationale for the proposed project is that knowledge of the biochemistry and overall biology of the DAHP synthases of P. aeruginosa will provide alternative approaches to combat this pathogen, especially as it pertains to the production of aromatic virulence factors. Two specific aims are proposed. In Specific Aim 1, each DAHP synthase of P. aeruginosa will be characterized in vitro to determine its catalytic properties, while the expression levels and patterns of the DAHP synthases will be measured using fluorescent-based reporters. A blueprint or layout for understanding DAHP synthase activity in P. aeruginosa pathogenesis will be generated. In Specific Aim 2, a series of DAHP synthase gene-deletion mutants will be constructed and examined for the production of aromatic virulence factors in vivo. A metabolomics approach will also be performed to measure the contribution of both nutritional factors and DAHP synthases regarding carbon flow or flux into the biosynthesis of DAHP and aromatic virulence factors. The approach is innovative because it addresses the biological significance of the one unifying cellular reaction that controls the production of all aromatic virulence factors in P. aeruginosa. The proposed project is significant, because knowledge of this reaction will provide fundamental insight into the cellular mechanisms and factors that actually dictate the quantities of aromatic virulence factors produced by this pathogen. Ultimately, such knowledge has the potential to identify new therapeutic targets to interfere with aromatic virulence factor production in P. aeruginosa. Project Number: 1R15AI188433-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Benjamin Lundgren | Institution: EASTERN WASHINGTON UNIVERSITY, CHENEY, WA | Award Amount: $470,978 | Activity Code: R15 | Study Section: Special Emphasis Panel[ZRG1 MGG-J (80)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R15AI18843301A1