Characterization of the Glyoxylate Pathway in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa infections are the leading cause of lung dysfunction and mortality in Cystic Fibrosis (CF) patients. Adaptation of P. aeruginosa to become a chronic pathogen of the CF lung includes acquisition of mutations that facilitate the prolonged survival of the bacterium in that environment. Thus in order to understand the adaptation strategies of the bacterium, it is imperative to study and characterize the actual CF isolates in addition to the non-CF isolates of P. aeruginosa. To identify chronic infection mechanisms of P. aeruginosa CF isolates, the Silo-Suh lab isolated transposon insertion mutants of a typical CF isolate strain, FRD1, that were decreased in virulence in an alfalfa seedling model of infection. One of the mutants contained a transposon insertion in aceA which encodes for isocitrate lyase, one of two enzymes involved in the glyoxylate pathway. The focus of my research was to characterize the expression of aceA and activity of isocitrate lyase to determine whether preferential utilization of the glyoxylate pathway is a part of the adaptation strategy for P. aeruginosa in the CF lung. I determined that the expression of the aceA gene is deregulated in the CF isolate FRD1 compared to the non-CF isolate PAO1. Moreover, deregulation of aceA appears to be common to other CF isolates of P. aeruginosa, suggesting this phenotype is important for adaptation of P. aeruginosa in the lung. In an effort to elucidate the molecular mechanism of aceA deregulation in FRD1, I discovered that RpoN, an alternative sigma factor, negatively regulates aceA expression in PAO1. This is a unique role for this

sigma factor. The exact mechanism by which aceA expression is deregulated by RpoN in FRD1 is unclear since this sigma factor appears to be active in FRD1. Thus it is likely that during adaptation, P. aeruginosa acquires a knockout mutation in a RpoN regulated gene whose role is to repress aceA expression in non-CF isolates. Finally, I determined that glcB, encoding for malate synthase, the second key enzyme of the glyoxylate pathway, is also required for virulence of P. aeruginosa on alfalfa and its expression is deregulated in FRD1 compared to PAO1. In addition, expression of a glcB is negatively regulated by RpoN in PAO1. This is the first study to systematically characterize expression of the glyoxylate pathway in P. aeruginosa and my data demonstrate the importance of this pathway for chronic infection isolates.