Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread

Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly i solated from the respiratory tract. Animal models of this infection are ext remely valuable for studies of virulence and immunity. We thus evaluated th e utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mic e following application of bacteria to the nares of anesthetized animals. B acterial aspiration into the lungs was rapid, and 67 to 100% of the inoculu m could be recovered within minutes from the lungs, with 0.1 to 1% of the i noculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin ex clusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect o n epithelial cells in vitro and virulence in animal models. Insertional mut ations in the exoU gene confer a noncytotoxic phenotype on mutant strains a nd decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the exoU gene into noncytotoxic strains o f P. aeruginosa lacking this gene affected virulence. Seven phenotypically noncytotoxic P. aeruginosa strains were transformed with pUCP19exoUspcU whi ch carries the exoU gene and its associated chaperone, Three of these strai ns became cytotoxic to cultured epithelial cells in vitro. These strains al l secreted ExoU, as confirmed by detection of the ExoU protein with specifi c antisera, The 50% lethal dose of exoU-expressing strains was significantl y lower for all three P. aeruginosa isolates carrying plasmid pUCP19exoUspc U than for the isogenic exoU-negative strains. mRNA specific for ExoU was r eadily detected in the lungs of animals infected with the transformed P. ae ruginosa strains. Introduction of the exoU gene confers a cytotoxic phenoty pe on some, but not all, otherwise-noncytotoxic P. aeruginosa strains and, for recombinant strains that could express ExoU, there was markedly increas ed virulence in a murine model of acute pneumonia and systemic spread.