Elucidation of the mechanism by which catecholamine stress hormones liberate iron from the innate immune defense proteins transferrin and lactoferrin

Abstract

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

FIG. 1.

Transferrin-NE interactions. (A) EPR spectra of 60 μM holo-Tf (equivalent to 120 μM total iron) in the absence and presence of increasing concentrations of NE. (B and D) Urea gels showing the time course of NE-mediated iron removal of 20 μM Tf incubated with 4 mM NE in the absence (B) and presence (D) of an Fe(II) sink (0.4 mM ferrozine); 40 μg of Tf was loaded per gel track. The numbers at the top of each gel show the length of incubation (minutes). Partially iron-saturated Tf (C-mono and N-mono), iron-saturated holo-Tf, and iron-free apo-Tf were used as protein markers. (C) EPR spectrum of 120 μM inorganic Fe(III) in the absence and presence of 12 mM NE ± 0.4 mM ferrozine compared with a similar concentration of an Fe(II) salt. Note that the Fe salts and NE alone produced no EPR spectrum, as indicated by the linear trace on the EPR profile shown. (E) Time course of Fe(II) production from 20 μM Tf incubated with 4 mM NE in the absence (−, squares) and presence (+, circles) of an Fe(II) sink (0.4 mM ferrozine). Monitoring of ferrozine-Fe(II) complex formation was done using a Varioskan densitometer set at 560 nm.

FIG. 2.

Tf-Epi and Tf-Dop interactions. (A) EPR spectrum of 60 μM holo-Tf in the absence and presence of increasing concentrations of Epi. (B and C) Urea gels showing the time course of iron removal of 20 μM Tf incubated with 4 mM Epi in the absence (B) and presence (C) of an Fe(II) sink (0.4 mM ferrozine); the numbers at the top of each gel show the length of incubation (minutes). (D) Time course of Fe(II) production from 20 μM Tf incubated with 4 mM Epi in the absence (−, squares) and presence (+, circles) of an Fe(II) sink (0.4 mM ferrozine). Monitoring of ferrozine-Fe(II) complex formation was done using a Varioskan densitometer set at 560 nm. (E) EPR spectrum of 60 μM holo-Tf in the presence of 12 mM Dop alone or in the presence of an Fe(II) sink (0.4 mM ferrozine) (12 mM +). (F) Time course of Fe(II) production from 20 μM Tf incubated with 4 mM Dop in the absence (squares) and presence (circles) of 0.4 mM ferrozine. (G and H) Urea gels showing the time course of iron removal of 20 μM Tf incubated with 4 mM Dop in the absence (G) and presence (H) of an Fe(II) sink (0.4 mM ferrozine).

FIG. 3.

Lf-NE interactions. (A) EPR spectrum of 60 μM Lf in the presence of increasing concentrations of NE. The symbol + indicates the spectra of Tf incubated with NE and 0.4 mM ferrozine. (B) Time course of Fe(II) production from 20 μM iron-saturated Lf incubated with 4 mM NE in the absence (−, squares) and presence (+, circles) of 0.4 mM ferrozine.

FIG. 4.

Human serum-Tf interactions with NE and Dop. (A) Freshly isolated whole sera from healthy volunteers were incubated for 72 h at 37°C with no additions (Control, which comprised the same volume of solvent used for the catecholamines) or with 10 μM additions of NE or Dop. (B) Comparative growth levels of an inoculum of 10² CFU/ml Staphylococcus epidermidis in the serum samples shown in panel A after 18 h of incubation at 37°C in a humidified static 5% CO₂ incubator; bacteria were enumerated as described in Materials and Methods. The results shown are the combined data from 3 separate growth analyses ± standard deviations.

FIG. 5.

NE can act as a bacterial pseudosiderophore. The models show the similarity of the structures of norepinephrine-Fe and enterobactin-Fe complexes.