The Bordetella bfe system: growth and transcriptional response to siderophores, catechols, and neuroendocrine catecholamines

Abstract

Ferric enterobactin utilization by Bordetella bronchiseptica and Bordetella pertussis requires the BfeA outer membrane receptor. Under iron-depleted growth conditions, transcription of bfeA is activated by the BfeR regulator by a mechanism requiring the siderophore enterobactin. In this study, enterobactin-inducible bfeA transcription was shown to be TonB independent. To determine whether other siderophores or nonsiderophore catechols could be utilized by the Bfe system, various compounds were tested for the abilities to promote the growth of iron-starved B. bronchiseptica and induce bfeA transcription. The BfeA receptor transported ferric salmochelin, corynebactin, and the synthetic siderophores TRENCAM and MECAM. Salmochelin and MECAM induced bfeA transcription in iron-starved Bordetella cells, but induction by corynebactin and TRENCAM was minimal. The neuroendocrine catecholamines epinephrine, norepinephrine, and dopamine exhibited a remarkable capacity to induce transcription of bfeA. Norepinephrine treatment of B. bronchiseptica resulted in BfeR-dependent bfeA transcription, elevated BfeA receptor production, and growth stimulation. Pyrocatechol, carbidopa, and isoproterenol were similarly strong inducers of bfeA transcription, whereas tyramine and 3,4-dihydroxymandelic acid demonstrated low inducing activity. The results indicate that the inducer structure requires a catechol group for function and that the ability to induce bfeA transcription does not necessarily correlate with the ability to stimulate bacterial growth. The expanded range of catechol siderophores transported by the BfeA receptor demonstrates the potential versatility of the Bordetella Bfe iron retrieval system. The finding that catecholamine neurotransmitters activate bfeA transcription and promote growth suggests that Bordetella cells can perceive and may benefit from neuroendocrine catecholamines on the respiratory epithelium.

FIG. 1.

TonB-independent induction of bfeA transcription in B. bronchiseptica. Wild-type B013N and BRM31 (tonB::pSSt2) cells harboring the bfeA-lacZ reporter fusion plasmid pMP3 or the promoterless lacZ vector control plasmid pMP220 were grown in SS medium and assayed for β-galactosidase activity as described in Materials and Methods. Genetic complementation of BRM31 was accomplished using the tonB⁺ exbBD⁺ plasmid pBB41, and pBBR1MCS-1 served as the vector control. Error bars, ±1 standard deviation from the means of triplicate cultures. Culture conditions were as follows: iron replete (solid bars), iron depleted (shaded bars), and iron depleted and supplemented with 3.3 μM enterobactin (open bars).

FIG. 2.

Molecular structures of catechol compounds (A) and of catecholamine synthesis pathway molecules and sympathetic nervous system agonists and antagonists (B).

FIG. 3.

Growth stimulation of B. bronchiseptica cells by natural and synthetic catechol siderophores. Wild-type B013N cells were seeded into iron-restricted agar as described in Materials and Methods and were provided with siderophores at 25 μM (solid bars), 12.5 μM (shaded bars), or 6.3 μM (open bars). Growth stimulation zones represent the means of triplicate bioassays and include the 6-mm diameter of the sample wells.

FIG. 4.

Norepinephrine-induced production of BfeA in B. bronchiseptica. Bacterial lysates from wild-type B013N cells grown in iron-depleted SS medium (−Fe) or in iron-depleted medium supplemented with 50 μM norepinephrine (−Fe+NE) were subjected to immunoblot analysis using a FepA-specific antiserum. The sizes of molecular mass standards (in kilodaltons) are shown on the left. Arrowhead indicates the immunoreactive BfeA protein.

FIG. 5.

Norepinephrine-induced bfeA transcription. Strains B013N (wild type) and BRM24 (ΔbfeR) carried the bfeA-lacZ reporter plasmid pMP3 or the vector control plasmid pMP220. Strain BRM24 was complemented by using the bfeR⁺ bfeA-lacZ plasmid pMP4. Bacteria were assayed for β-galactosidase activity after growth in SS medium under the following conditions: iron replete (solid bars), iron replete and supplemented with 50 μM norepinephrine (shaded bars), iron depleted (striped bars), or iron depleted and supplemented with 50 μM norepinephrine (open bars). Error bars, ±1 standard deviation from the means of triplicate cultures.

FIG. 6.

Growth stimulation by norepinephrine. B. bronchiseptica cells were cultured in iron-depleted SS medium (SS) or iron-depleted SS medium containing 30% FBS (SS+FBS), and the growth yield after 20 h was measured densitometrically at a wavelength of 600 nm. Cultures either had no supplementation (solid bars) or were supplemented with either enterobactin (shaded bars), norepinephrine (striped bars), or both enterobactin and norepinephrine (open bars).

FIG. 7.

Analysis of bfeA transcriptional activation by catecholamines. B. bronchiseptica B013N carrying the bfeA-lacZ reporter plasmid pMP3 was assayed for β-galactosidase activity as described in Materials and Methods. Bacteria were grown in iron-depleted SS medium and supplemented with one of the following catechol compounds: enterobactin (solid squares), norepinephrine (open squares), epinephrine (solid triangles), dopamine (open circles), l-DOPA (open triangles), and tyrosine (solid circles). Values represent means of triplicate cultures ± 1 standard deviation. Error bars representing less than ±50 Miller units are not shown.