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Comparative Study
. 2000 Jul;68(7):3998-4004.
doi: 10.1128/IAI.68.7.3998-4004.2000.

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

M Allewelt  1 , F T Coleman, M Grout, G P Priebe, G B Pier
Affiliations
Comparative Study

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

M Allewelt et al. Infect Immun. 2000 Jul.

Abstract

Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the exoU gene confer a noncytotoxic phenotype on mutant strains and decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the exoU gene into noncytotoxic strains of P. aeruginosa lacking this gene affected virulence. Seven phenotypically noncytotoxic P. aeruginosa strains were transformed with pUCP19exoUspcU which carries the exoU gene and its associated chaperone. Three of these strains became cytotoxic to cultured epithelial cells in vitro. These strains all secreted ExoU, as confirmed by detection of the ExoU protein with specific antisera. The 50% lethal dose of exoU-expressing strains was significantly lower for all three P. aeruginosa isolates carrying plasmid pUCP19exoUspcU than for the isogenic exoU-negative strains. mRNA specific for ExoU was readily detected in the lungs of animals infected with the transformed P. aeruginosa strains. Introduction of the exoU gene confers a cytotoxic phenotype on some, but not all, otherwise-noncytotoxic P. aeruginosa strains and, for recombinant strains that could express ExoU, there was markedly increased virulence in a murine model of acute pneumonia and systemic spread.

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Figures

FIG. 1
FIG. 1
Rapid aspiration of four different P. aeruginosa strains from the nares to the lungs after application of the indicated inoculum in a 20-μl volume to the nares of anesthetized mice. Bars represent the mean, and the error bars show the standard deviations. The percentage of the inoculum that was recovered from the lungs is indicated above each pair of bars.
FIG. 2
FIG. 2
Comparison of total and internalized (resistant to gentamicin in single cell suspensions of lung) noncytotoxic P. aeruginosa PAO1 (left) and cytotoxic P. aeruginosa 6077 (right) cells at the indicated times after application to the nares of anesthetized mice. Bars represent the mean CFU, and the error bars show the standard deviations. The inoculum for P. aeruginosa PAO1 was 3 × 108 CFU/nose; the inoculum for P. aeruginosa 6077 was 3 × 106 CFU/nose.
FIG. 3
FIG. 3
Agarose gel stained with ethidium bromide showing the presence or absence of the 428-bp amplified exoU gene fragment in 14 clinical isolates of P. aeruginosa. Lanes: MW, molecular weight marker, 1, positive control pUCP19exoUspcU; 2, strain 45203; 3, strain 9156; 4, strain 56184; 5, strain Weaver; 6, strain 15921; 7, strain 1597; 8, strain Becker; 9, strain 29185; 10, strain 9882; 11, strain Rhodes; 12, strain 1947; 13, strain 9326; 14, strain 05074; 15, strain 3006.
FIG. 4
FIG. 4
Immuno-dot blot of expression of recombinant ExoU protein in culture supernates of P. aeruginosa PAO1, 15921, and PAO6ad carrying either the control, vector plasmid pUCP19 (Vec), or the plasmid containing the exoU gene, pUCP19exoUspcU (ExoU).
FIG. 5
FIG. 5
Comparison of CFU/gram of lung tissue 14 to 18 h after intranasal infection of anesthetized mice with isogenic P. aeruginosa PAO1(pUCP19) or PAO1(pUCP19exoUspcU). Bars indicate the means, and the error bars show the standard deviations. P values were determined by unpaired Student t tests.
FIG. 6
FIG. 6
Demonstration of elaboration of mRNA for ExoU in infected lung tissue of mice. RT-PCR of lung tissue from mice infected with either the exoU+ PAO1(pUCP19exoUspcU) strain or the parental strain carrying the cloning vector, PAO1(pUCP19). mRNA from tissue was reverse transcribed and amplified with primers specific to the rpoB gene to yield a product of 314 bp or with primers specific to the exoU gene to yield a product of 428 bp. Molecular weight markers on the left are oligonucleotides differing by 100 bp.
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