Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice

Abstract

Pseudomonas aeruginosa produces siderophores, pyoverdin and pyochelin, for high-affinity iron uptake. To investigate their contribution to P. aeruginosa infections, we constructed allelic exchange mutants from strain PAO1 which were deficient in producing one or both of the siderophores. When inoculated into the calf muscles of immunosuppressed mice, pyochelin-deficient and pyoverdin-deficient mutants grew and killed the animals as efficiently as PAO1. In contrast, the pyochelin- and pyoverdin-deficient (double) mutant did not show lethal virulence, although it did infect the muscles. On the other hand, when inoculated intranasally, all mutants grew in the lungs and killed immunosuppressed mice. Compared with PAO1, however, the pyoverdin-deficient mutant and the double mutant grew poorly in the lungs, and the latter was significantly attenuated for virulence. Irrespective of the inoculation route, the pyoverdin-deficient and doubly deficient mutants detected in the blood were significantly less numerous than PAO1. Additionally, in vitro examination demonstrated that the growth of the double mutant was extremely reduced under a free-iron-restricted condition with apotransferrin but that the growth reduction was completely canceled by supplementation with hemoglobin as a heme source. These results suggest that both pyoverdin and pyochelin are required for efficient bacterial growth and full expression of virulence in P. aeruginosa infection, although pyoverdin may be comparatively more important for bacterial growth and dissemination. However, the siderophores were not always required for infection. It is possible that non-siderophore-mediated iron acquisition, such as via heme uptake, might also play an important role in P. aeruginosa infections.

FIG. 1

Growth (A) and production of siderophores Pch (B) and Pvd (C) by P. aeruginosa strain PAO1 and its allelic exchange mutants in SMMCA supplemented with 1 μM FeCl₃. A bacterial growth assay was performed in a 96-well plate (0.1 ml of culture/well), and the growth was measured as OD₅₉₀ in a SPECTRAmax (Molecular Devices, Sunnyvale, Calif.), which is a microplate spectrophotometer with incubation and mixing functions, after bacterial inoculations at approximately 10⁶ CFU/ml. ○, strain PAO1; ▵, the pchD-inactivated mutant, PAD02; □, the pvdA-inactivated mutant, PAD06; +, the pchD- and pvdA-inactivated mutant, PAD07; ×, no inoculum (medium control). Pch (B) and Pvd (C) secreted into the culture supernatant were fluorometrically or photometrically measured as described in Materials and Methods; the values for Pch and Pvd amounts (fluorometric measurement with excitation at 350 nm and emission at 440 nm and OD₄₀₅, respectively) were corrected by dividing by the values for bacterial growth (OD₅₉₀).

FIG. 2

Effects of apoTsf on the growth of P. aeruginosa strain PAO1 and its siderophore production mutants in SMMCA containing 10 μM FeCl₃ and 20 mM sodium bicarbonate (A), and effects of supplementation with FeSO₄ (B) or hemoglobin (C) on their growth in the medium including 25 μM apoTsf. Growth was measured as the end point OD₅₉₀ of culture 20 h after bacterial inoculation. ○, strain PAO1; ▵, the Pch-deficient mutant, PAD02; □, the Pvd-deficient mutant, PAD06; ●, the Pch- and Pvd-deficient mutant, PAD07; ×, no inoculum (medium control).