Discovering the enzyme(s) that catalyze the reduction of Phenazine-1-Carboxylic Acid (PCA) in Pseudomonas aeruginosa
Authors:Nate Glasser, Ben Wang
Mentor:Dianne Newman, Professor of Biology and Geobiology; Investigator, Howard Hughes Medical Institute, California Institute of Technology (Caltech)
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and is most well-known for causing lethal lung infections in persons with cystic fibrosis (CF). These bacteria synthesize colorful redox-active compounds called phenazines that not only serve as virulent factors but also directly enable the survival of the producing organism. Phenazines are often found in a reduced state when carrying out their various functions; however the enzyme that catalyzes the reduction of phenazines is currently unknown. One such phenazine is called Phenazine-1-Carboxylic Acid (PCA), which changes from clear to yellow when it becomes reduced. The purpose of this project was to identify the enzyme(s) that catalyze the reduction of PCA. Two approaches were used to identify this enzyme. First, a “blind purification” was performed on P. aeruginosa lysate in which soluble proteins were purified through successive columns and assayed for PCA reduction activity. Second, based on previous research it was thought that PCA reduction could be coupled to the TCA cycle in P. aeruginosa; in this approach different components of the TCA cycle were assayed for PCA reduction activity. So far, experiments have shown that P. aeruginosa lysate reduces PCA in the presence of NADH, α-ketoglutarate, and pyruvate. Dihydrolipoamide dehydrogenase (DLDH) is found in both the α-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes and is also a redox-active NADH binding protein. Thus, the current hypothesis is that DLDH is the enzyme that catalyzes the reduction of PCA. This study has shown that DLDH indeed catalyzes the reduction of PCA in vitro. Furthermore, mutants deficient in the production of DLDH are currently being generated to test if DLDH also catalyzes the reduction of PCA in vivo. As phenazines are important virulence factors for P. aeruginosa, understanding the reduction pathway of phenazines can provide a novel avenue for treatments against the pathogen in chronic infections.