Norepinephrine mediates acquisition of transferrin-iron in Bordetella bronchiseptica

Abstract

Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of Bordetella bronchiseptica in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the Bordetella bfeA enterobactin catechol xenosiderophore receptor gene, neither a bfeA mutant nor a bfeR regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a tonB mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The B. bronchiseptica genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of (55)Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates B. bronchiseptica iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.

FIG. 1.

Analysis of Bordetella iron acquisition via norepinephrine and siderophores. B. bronchiseptica ΔalcA strain BRM26 was cultured in iron-depleted SS medium (A), iron-depleted medium containing 200 μg/ml transferrin (B), or iron-depleted medium containing 200 μg/ml lactoferrin (C). Bacteria were inoculated at an initial density of ∼100 CFU/ml, and growth was measured densitometrically. Parallel cultures were additionally supplemented as follows: 36 μM FeSO₄ (diamonds), 50 μM norepinephrine and 1 μM enterobactin (circles), 1 μM enterobactin (triangles), 50 μM norepinephrine (squares), and no supplementation (asterisks).

FIG. 2.

Growth stimulation by norepinephrine is independent of the BfeA enterobactin receptor and BfeR regulator proteins. (A) B. bronchiseptica strains B013N (bfeA⁺) and BRM33 (ΔbfeA) and (B) strains B013N (bfeR⁺) and BRM24 (ΔbfeR) were used. B. bronchiseptica cells were grown in iron-replete medium (solid bars) or iron-depleted medium supplemented with either 200 μg/ml transferrin (shaded bars) or 200 μg/ml transferrin and 50 μM norepinephrine (open bars). Growth yields were measured densitometrically after 40 h of incubation. Values represent means ± 1 standard deviation (error bars) from triplicate cultures.

FIG. 3.

TonB-dependent growth stimulation by norepinephrine. B. bronchiseptica strains B013N (tonB⁺), BRM31(pBBR1MCS-1) (tonB::pSSt2), and BRM31(pBB41) (tonB::pSSt2/tonB⁺) were used. B. bronchiseptica cells were grown in iron-replete medium (solid bars) or iron-depleted medium supplemented with either 200 μg/ml transferrin (shaded bars) or 200 μg/ml transferrin and 50 μM norepinephrine (open bars). Growth yields were measured densitometrically after 40 h of incubation. Values represent means ± 1 standard deviation (error bars) from triplicate cultures.

FIG. 4.

TonB-dependent cell association of ⁵⁵Fe and [³H]norepinephrine. (A) B. bronchiseptica strains B013N (tonB⁺), BRM31(pBBR1MCS- 1) (tonB::pSSt2), and BRM31(pBB41) (tonB::pSSt2/tonB⁺) were used. Bacteria were grown in iron-depleted medium containing 200 μg/ml ⁵⁵Fe-transferrin (solid bars), 200 μg/ml ⁵⁵Fe-transferrin plus 50 μM norepinephrine (shaded bars), or 200 μg/ml ⁵⁵Fe-transferrin plus 50 μM norepinephrine plus 36 μM FeSO₄ (open bars). (B) B. bronchiseptica strains B013N (solid bars), BRM31(pBBR1MCS-1) (shaded bars), and BRM31(pBB41) (open bars) were used. Iron-depleted basal medium containing [³H]norepinephrine was supplemented with either 200 μg/ml transferrin plus 50 μM norepinephrine (FeTf + NE) or 200 μM transferrin plus 50 μg/ml norepinephrine plus 36 μM FeSO₄ (FeTF + NE + FeSO₄). Cell-associated levels of ⁵⁵Fe or [³H]norepinephrine were determined by scintillation counting and normalized based on the optical density at 600 nm (OD₆₀₀) after 24 h of incubation. Values represent means ± 1 standard deviation (error bars) from triplicate determinations.