Pseudomonas aeruginosa pyocyanin is critical for lung infection in mice

Abstract

Pseudomonas aeruginosa secretes copious amounts of the redox-active phenazine, pyocyanin (PCN), during cystic fibrosis lung infection. PCN has been shown to interfere with a variety of cellular processes in cultured lung epithelial cells. Here, by using two respiratory tract models of infection, we demonstrate that PCN mediates tissue damage and necrosis during lung infection.

FIG. 1.

PCN contributes to pneumonia development in adult mouse lungs. (A) PCN causes neutrophil influx and small-airway congestion. Histological sections from 16 h postinfection are shown. (B and C) PCN-deficient P. aeruginosa mutants are less able to cause pneumonia. Adult CD-1 mice were infected with 10⁷ cells of PA14 and its isogenic PCN mutants, the phzB1 and mvfR mutants (B), or PAO1 and its isogenic PCN-deficient mutants, the phzM and phzS mutants (C). Infected lungs were collected at 16 h postinfection, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. Original magnification, ×10.

FIG. 2.

PCN-deficient P. aeruginosa mutants have impaired virulence in an acute pneumonia model of mouse infection. (A and B) PCN-deficient mutants of P. aeruginosa are highly attenuated in single respiratory tract infections. CD-1 mice (six per group) were infected intranasally with wild-type PA14 or the mvfR or phzB1 isogenic PCN-deficient mutant (A) and with wild-type PAO1 or the phzM or phzS isogenic PCN-deficient mutant (B). Attenuation is defined as the log₁₀ difference in CFU between the wild type and mutant bacteria recovered from lung tissue 16 h after inoculation. The means ± standard errors of results from six mice are shown. P values for results: mvfR mutant, 0.040; phzB1 mutant, 0.036; phzM mutant, 0.003; phzS mutant, 0.013. (C) The competitive index is defined as the output ratio of the numbers of mutant to wild-type bacteria divided by the input ratio of the numbers of mutant to wild-type bacteria. Thus, if a mutant strain is as competitive as its isogenic wild-type parent, a value of 1 will be achieved, indicating that the mutant is not attenuated. The means ± standard errors of results from five mice are shown. P values for results: phzB1 mutant, 0.002; mvfR mutant, 0.006; phzM mutant, 0.007; phzS mutant, 0.007.

FIG. 3.

Coadministration of PCN rescues the virulence of PCN-deficient mutants in mouse lungs. CD-1 mice (five per group) were coinstilled intranasally with 50 μg of PCN and 10⁶ bacterial cells of wild-type PA14 or its mvfR or phzB1 isogenic PCN-deficient mutant (A) or with the wild-type PAO1 or its isogenic phzM or phzS PCN-deficient mutant (B). Attenuation is defined as the log₁₀ difference in CFU between wild-type and mutant bacteria recovered from lung tissue 16 h after inoculation. The means ± standard errors of results from five mice are shown. P values for results: mvfR mutant, 0.002; phzB1 mutant, 0.167; phzM mutant, 0.618; phzS mutant, 0.876.

FIG. 4.

PCN-deficient mutants are less competitive than wild-type P. aeruginosa in a mouse chronic lung infection model. In vivo competition assays between the wild type and individual PCN mutants embedded in agar beads were carried out by infecting CD-1 mice (five per group) intranasally with a 1:1 ratio of wild-type PA14 and one of its isogenic PCN mutants, either the phzB1 or the mvfR mutant, or PAO1 and one of its isogenic PCN-deficient mutants, either the phzM or the phzS mutant. Infected lungs were recovered 60 h after infection for bacterial load determination. The means ± standard errors of results from five mice are shown. P values for results: mvfR mutant, 1.9 × 10⁻⁵; phzB1 mutant, 0.002; phzM mutant, 0.007; phzS mutant, 0.007.