Insights into mechanisms of Pseudomonas aeruginosa virulence: cyanide as a weapon and the complexity of its regulation

Abstract

This dissertation presents investigations into virulence mechanisms and virulence-gene regulatory circuitry in the bacterium Pseudomonas aeruginosa . P. aeruginosa is a medically important human pathogen that causes serious disease in the lungs of cystic fibrosis patients. It also is virulent towards fungi, nematodes, plants and insects. To investigate virulence mechanisms employed by the bacterium, a model pathogenesis system between P. aeruginosa PAO1 and Caenorhabditis elegans was investigated. PAO1 rapidly paralyzes and kills C. elegans by producing a diffusible poison or toxin that requires an intact copy of the C. elegans gene egl-9. Chapter 2 presents experiments that identify hydrogen cyanide as the sole or primary poison responsible for this killing. A genetic screen for killing-defective mutants, an analysis of the genes identified, and a reconstruction of the killing phenomenon showed that hydrogen cyanide is both necessary and sufficient to effect paralytic killing in an egl-9-dependent manner. Many virulence determinants in P. aeruginosa are controlled by a complex hierarchy of interacting regulatory factors. Chapter 3 presents a screen for mutants of PAO1 with defects in pyocyanin production. The screen resulted in the identification of four genes responsible for production of a small-molecule regulatory factor, the Pseudomonas quinolone signal (PQS). Transcription of quorum-sensing-controlled genes on the chromosome was also examined in mutant strains affected in pyocyanin production. The results revealed new hierarchical interactions between different components of the regulatory circuitry. Chapter 4 presents three additional findings: construction and analysis of a transposon tool (ISphoA/hah-Tc) for use in P. aeruginosa, analysis of transcription at the hydrogen cyanide biosynthetic locus ( hcnABC) in mutant strains that don't produce cyanide, and evidence that egl-9 worms resist cyanide poisoning by enhancing mitochondrial respiration and glycolysis. In conclusion, these studies have elucidated both sides of a virulence/resistance interaction between P. aeruginosa and C. elegans, and have uncovered new genes and genetic interactions in the virulence-gene regulatory circuitry in P. aeruginosa.