Elucidation of the Mechanism by Which Catecholamine Stress Hormones Liberate Iron from the Innate Immune Defense Proteins Transferrin and Lactoferrin

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.Iron is a key nutritional element required for the growth of almost all bacteria (15, 22); therefore, its sequestration by the mammalian ferric-iron-binding proteins (principally transferrin Tf] in serum and lactoferrin Lf] in mucosal secretions) represents a primary nonspecific host defense mechanism against microbial infection. Tf has one of the highest metal binding affinities recorded, with a binding constant for ferric iron of 10⁻²³ M (16). The principal physiological role of serum Tf is Fe transport through the circulating blood and its release to Fe-dependent cells; its concentration in serum is usually about 35 μM (16). Importantly, serum Tf is not iron replete, with about 70% of it existing in the apo form (16). Work in our laboratories has shown that the “fight or flight” catecholamine stress hormones epinephrine (Epi), norepinephrine (NE), and dopamine (Dop) and the widely used structurally similar inotropes (heart and kidney therapeutic drugs) isoprenaline and dobutamine are all able to form complexes with Tf and Lf (7, 8, 10, 21). This complex formation is important microbiologically, as it reduces the Fe-binding capability of these key innate immune defense proteins to an almost insignificant level and renders them vulnerable to Fe theft by bacterial pathogens that would be unable to access this normally highly secure iron. We and others have shown that these catecholamines are all able to support greater-than-millionfold increases in bacterial growth by providing iron from Tf (1, 7, 8, 10, 11, 21). Significantly, in terms of their ability to deliver Tf/Lf-complexed iron to bacteria, certain pharmacologically inactive catechol-containing metabolites were also found to be similar in potency and effect to the parent catecholamine molecule (8).The interaction between catecholamines, Tf, and Lf can reduce the bacteriostatic nature of blood and serum and mucosal secretions to the extent that they become a highly supportive bacterial culture medium (7, 8, 10, 11, 21). This ability of stress hormones to mediate bacterial acquisition of Tf/Lf-iron has been shown to have important clinical implications; for example, they have been proposed to have roles in sepsis due to the formation of staphylococcal biofilms in intravenous lines (18) and in the development of stress-related intra-abdominal sepsis by Gram-negative bacteria (8). Although we and others have identified some of the molecular components that bacteria use to acquire iron from these stress hormone-Tf/Lf complexes (1, 4, 7, 9, 25), the precise mechanism(s) by which the catecholamines themselves modulate Tf and Lf iron binding remain to be determined. Elucidation of the mechanism by which stress-elaborated hormones enable bacterial-pathogen access to host-sequestered iron is therefore important both scientifically and clinically. Because the iron within Tf and Lf is in a high-spin Fe(III) oxidation state (16) and therefore paramagnetic, electron paramagnetic resonance (EPR) spectrometry is an ideal tool to study the dynamics of the interaction between the catecholamines and Tf and Lf. The present study utilized EPR spectrometry, biochemical, and microbiological approaches to elucidate the mechanism by which catecholamine stress hormones and inotropes liberate Tf- and Lf-complexed Fe.