The Pseudomonas aeruginosa pyochelin-iron uptake pathway and its metal specificity

Abstract

Pyochelin (Pch) is one of the two major siderophores produced and secreted by Pseudomonas aeruginosa PAO1 to assimilate iron. It chelates iron in the extracellular medium and transports it into the cell via a specific outer membrane transporter, FptA. We used the fluorescent properties of Pch to show that this siderophore chelates, in addition to Fe(3+) albeit with substantially lower affinities, Ag(+), Al(3+), Cd(2+), Co(2+), Cr(2+), Cu(2+), Eu(3+), Ga(3+), Hg(2+), Mn(2+), Ni(2+), Pb(2+), Sn(2+), Tb(3+), Tl(+), and Zn(2+). Surprisingly, the Pch complexes with all these metals bound to FptA with affinities in the range of 10 nM to 4.8 microM (the affinity of Pch-Fe is 10 nM) and were able to inhibit, with various efficiencies, Pch-(55)Fe uptake in vivo. We used inductively coupled plasma atomic emission spectrometry to follow metal uptake by P. aeruginosa. Energy-dependent metal uptake, in the presence of Pch, was efficient only for Fe(3+). Co(2+), Ga(3+), and Ni(2+) were also transported, but the uptake rates were 23- to 35-fold lower than that for Fe(3+). No uptake was seen for all the other metals. Thus, cell surface FptA has broad metal specificity at the binding stage but is much more selective for the metal uptake process. This uptake pathway does not appear to efficiently assimilate any metal other than Fe(3+).

FIG. 1.

(A) Variation of Pch fluorescence in the presence of various metal ions. The fluorescence at 430 nm (excitation wavelength, 347 nm) of metal-free Pch was taken as the reference (100%). Error bars indicate standard deviations. (B) Fluorescence spectra of Pch incubated in the presence of Ag⁺ (▵), Al³⁺ (▾) Ga³⁺ (⋄), Fe³⁺ (○), Hg³⁺ (▪), and Sn²⁺ (•). The fluorescence spectrum of metal-free Pch is shown as a black line. For both panels, 25 μM Pch was incubated overnight with 12.5 μM metals in 50 mM Tris-HCl (pH 7.0), and the fluorescence was then monitored (excitation wavelength, 347 nm). a.u., arbitrary units.

FIG. 2.

Ability of Pch-metal complexes to inhibit Pch-⁵⁵Fe uptake. Pvd- and Pch-deficient PAD07 cells at an OD₆₀₀ of 1 were incubated for 45 min in the presence of 100 nM Pch-⁵⁵Fe at 37°C. The mixtures were then filtered and radioactivity counted. The amount of ⁵⁵Fe transported in this experiment was used as a reference (defined as 100% uptake). This experiment was repeated in the absence of cells (bars labeled “Buffer”). The experiment was also repeated in the presence of cells and an excess of metal-free Pch and in the presence of cells and of each Pch-metal complex (black and white bars correspond to 1 and 10 μM metal, respectively). Pch-metal complexes were prepared by preincubating each metal in the presence of a 20-fold excess of Pch overnight. As a control, for each metal tested, the experiment was repeated in the absence of cells (data not shown). All the data are means and standard deviations from three independent experiments. The black line indicates the 100% of Pch-⁵⁵Fe uptake, and the gray line shows that most of the Pch-metal complexes inhibited 28% of the Pch-⁵⁵Fe uptake.

FIG. 3.

Competition by unlabeled Pch-metal for the binding of Pch-⁵⁵Fe to FptA in vivo. Experiments were carried out as described in Materials and Methods with 1 nM Pch-⁵⁵Fe, Pch- and Pvd-deficient PAD07 cells at an OD₆₀₀ of 0.6, incubation at 0°C, and various concentrations of metal-free Pch or of Pch-metal at Pch/metal ratios of 1:2, 2:1, and 1:10. The values reported are representative of two experiments, which gave similar results. These data were used to calculate the K_i values presented in Table 2.

FIG. 4.

Proton motive force-dependent metal incorporated into P. aeruginosa cells by the Pch uptake pathway. (A) Pvd- and Pch-deficient PAD07 cells at an OD₆₀₀ of 1 were incubated at 37°C with 5 μM of each Pch-metal complex in the presence (white bars) or absence (black bars) of 200 μM CCCP. After 45 min of incubation, the cells were harvested and washed and the metal content determined by ICP-AES. Pch-metal complexes were prepared by incubating each metal in the presence of a 20-fold excess of Pch overnight. The data are means and standard deviations from three independent experiments. (B) The experiment was repeated in parallel with PAD07 cells in the presence (white bars) or absence (black bars) of CCCP and with K2388 cells (ΔfptA and Pvd⁻) (grey bars) for Co²⁺, Ga³⁺, and Ni²⁺.

FIG. 5.

Role of the Pch pathway in P. aeruginosa metal toxicity. P. aeruginosa CDC5(pPVR2) (a strain unable to produce Pvd) (black bars) and PAD07 (a strain unable to produce both Pvd and Pch) (white bars) were grown overnight in the presence or absence of each metal ion (100 μM). After 20 h of culture, the OD₆₀₀ was determined. The culture in the absence of metal was used as the reference (100%). The data are means and standard deviations from triplicate experiments.