Hybrid pathogenicity island PAGI-5 contributes to the highly virulent phenotype of a Pseudomonas aeruginosa isolate in mammals

Abstract

Most known virulence determinants of Pseudomonas aeruginosa are remarkably conserved in this bacterium's core genome, yet individual strains differ significantly in virulence. One explanation for this discrepancy is that pathogenicity islands, regions of DNA found in some strains but not in others, contribute to the overall virulence of P. aeruginosa. Here we employed a strategy in which the virulence of a panel of P. aeruginosa isolates was tested in mouse and plant models of disease, and a highly virulent isolate, PSE9, was chosen for comparison by subtractive hybridization to a less virulent strain, PAO1. The resulting subtractive hybridization sequences were used as tags to identify genomic islands found in PSE9 but absent in PAO1. One 99-kb island, designated P. aeruginosa genomic island 5 (PAGI-5), was a hybrid of the known P. aeruginosa island PAPI-1 and novel sequences. Whereas the PAPI-1-like sequences were found in most tested isolates, the novel sequences were found only in the most virulent isolates. Deletional analysis confirmed that some of these novel sequences contributed to the highly virulent phenotype of PSE9. These results indicate that targeting highly virulent strains of P. aeruginosa may be a useful strategy for identifying pathogenicity islands and novel virulence determinants.

FIG. 1.

Virulence of P. aeruginosa isolates in a mouse model of acute pneumonia and distribution of PAGI-5 regions among these isolates. (A) Mice were infected with a range of bacterial inocula and monitored over the subsequent 7 days to calculate LD₅₀s. The y axis is inverted so that the results for more virulent isolates are indicated by taller bars. The gray bars indicate ExoU-secreting isolates, the diagonally striped bars indicate ExoS-secreting isolates, and the open bars indicate nonsecreting isolates. “Other” refers to isolate PSE7, which had a functional type III secretion system but did not secrete any effector proteins. (These results were previously published [51] and are reproduced here in adapted form.) (B) Presence of PAGI-5 conserved and novel sequences in the panel of P. aeruginosa clinical isolates. The regions of PAGI-5 are indicated on the left, and a plus sign indicates the presence of the sequence.

FIG. 2.

Virulence of P. aeruginosa isolates in a lettuce leaf model of infection. (A) Lettuce leaf infected with positive control strain PA14, clinical isolate PSE9, and reference strain PAO1. (B) Area of soft rot caused by each P. aeruginosa isolate normalized to the area of soft rot caused by PA14. The data are the means ± standard errors of the means for three inoculation sites on a single leaf. For an explanation of the type III secretion profiles see the legend to Fig. 1.

FIG. 3.

Flow chart showing the results of analysis of 75 PSE9 subtractive hybridization products. After removal of false positives, redundant clones, and sequences from previously characterized genomic islands, 22 distinct sequences remained. These sequences were used to screen a PSE9 genomic library.

FIG. 4.

Map of PAGI-5. Arrows represent ORFs and are oriented in the direction of transcription. Gray arrows represent ORFs with similarity to PAPI-1 sequences, and open arrows represent ORFs that lack PAPI-1 similarity. Black arrows represent PAO1 ORFs that flank PAGI-5. Diagonal stripes indicate ORFs that are predicted to encode proteins with sequences that do not suggest a function, and speckled arrows indicate ORFs expected to play a role in DNA mobility. tRNA attL and attR sites are indicated by vertical arrows. G+C contents are indicated above the ORFs and were calculated using a sliding 100-bp window. PAGI-5 ORFs are designated “5PGX,” where “X” is the sequential number of the ORF within the genomic island.

FIG. 5.

Alignment of PAGI-5, PAPI-1, and ExoU island A. Dark bands and ORFs represent conserved nucleotide sequences, whereas open ORFs indicate unrelated sequences. The double lines beneath PAGI-5 indicate the sequences that were amplified by PCR to detect the presence of the corresponding conserved and novel regions of PAGI-5 in the panel of 35 P. aeruginosa clinical isolates.

FIG. 6.

Survival of PSE9, PSE9ΔNR-I, PSE9ΔNR-II, and PAO1 in a mouse model of acute pneumonia. The symbols indicate the percentage of animals surviving in each experimental group over time. Each group contained 14 to 35 mice pooled from at least two separate experiments. An asterisk indicates that values are significantly different (P = 0.0036, log rank test).

FIG. 7.

Results of competition assays using mixtures of PSE9 and PAO1, PSE9ΔNR-I, or PSE9ΔNR-II at 22 h postinfection for the lungs and spleen. Data from eight or nine mice from two experiments were pooled. Each symbol indicates the CI for the tissue sample from one mouse, and the bars indicate medians. CIs for parental strain PSE9 in competition with either PSE9ΔNR-I, PSE9ΔNR-II, or PAO1 were compared to CIs for parental strain PSE9 in competition with PSE9 tagged with a gentamicin resistance cassette to determine whether differences were significant. Statistical significance was determined using a two-tailed unpaired Student's t test (*, P ≤ 0.05; **, P ≤ 0.005).