Determinants of Pseudomonas aeruginosa ocular virulence

Pseudomonas aeruginosa is a Gram-negative pathogen which causes severe eye disease. This study examined individual genes as well as systems of virulence gene regulation in the context of restoring pathogenicity to an avirulent strain. Isolated from a human eye, P. aeruginosa strain Paer1 caused minimal pathology. In contrast another human isolate, strain KEI 1025, destroyed the cornea. Strain Paer1 was compared to KEI 1025 on a genomic level through suppression subtractive hybridization. Differences between the two strain's genomes were identified, and examined for relevance to ocular virulence. An adenylate cyclase (CyaB), the Las quorum sensing system (LasR, RsaL, LasI), and a major protease of P. aeruginosa (LasB) were restored to Paer1. Other quorum sensing response modifiers (GacA and Vfr) were examined in Paer1 as well. Polyphosphate kinase (PPK1), a potential target for antimicrobial therapy, was examined for its role in corneal virulence and stress response. Protein secretion in strains of Paer1 and KEI 1025 grown in a simulated ocular environment was examined, as well as an analysis of mRNA induced in these strains during a corneal infection.;GacA, Vfr, CyaB were intact however LasB was not and this in part related to decreased virulence in Paer1. Like LasB, PPKI played a role in ocular virulence. Without PPK1, P. aeruginosa was notably susceptible to oxidative stress. When restored to Paer1, the LasR/LasI quorum sensing was induced in a rich environment, and repressed in a minimal environment. A novel Southern blot based approach to expression analysis was attempted, but it requires some refinement.;In conclusion, Paer1 was compared to KEI 1025 on multiple levels to assess differences that relate to ocular virulence. Paer1 lost a major mechanism of gene regulation (LasR/LasI) as well as an effector of that system (LasB). When LasR/LasI is restored to Paer1 the severity of the disease is lessened due to repression through an unknown mechanism. When LasB is restored to Paer1 the organism produces significantly more corneal pathology.