The growth response of Escherichia coli to neurotransmitters and related catecholamine drugs requires a functional enterobactin biosynthesis and uptake system - PubMed (original) (raw)

Claire L Burton et al. Infect Immun. 2002 Nov.

Abstract

The neurotransmitter norepinephrine (NE) stimulates the growth of low inocula of Escherichia coli in a minimal medium (SAPI) supplemented with serum (SAPI+serum) and induces the production of an "autoinducer" (AI) which, in turn, promotes E. coli growth in the absence of NE. Given the importance of NE, epinephrine, and their corresponding adrenergic agonists and antagonists in clinical medicine, we sought to investigate the molecular basis for these observations. Using a variety of NE precursors, metabolites, and therapeutic agents, we demonstrated that their ability to stimulate E. coli growth in SAPI+serum is dependent on the presence of a catechol (1,2-dihydroxybenzene) moiety with maximal activity requiring a two-carbon substituent incorporating a terminal primary amine. Serum contains the iron-binding glycoprotein, transferrin, and when SAPI+serum was supplemented with sufficient Fe(3+) to saturate transferrin, growth inhibition was relieved. Other metal cations, including Mg(2+), Ca(2+), and Zn(2+), had no effect. These data suggested that the stimulation of E. coli growth by NE in SAPI+serum may involve the catecholate siderophore, enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine. Consistent with this hypothesis, E. coli strains with mutations in ferrienterobactin transport (fepA or tonB) or enterobactin biosynthesis (entA) did not respond to NE. Furthermore, NE induced expression of the ferrienterobactin receptor, FepA, during growth in SAPI+serum. The enterobactin degradation product, 2,3-dihydroxybenzoylserine (DBS) was as effective as NE in stimulating the growth of E. coli and mutations in fepA or tonB abolished the DBS-dependent growth stimulation. In contrast to NE, however, DBS stimulated the growth of the entA mutant. Moreover, after growth in an iron-limited M9 medium in the absence of NE, ethyl acetate extracts of the E. coli entA(+) parent but not of the entA mutant contained AI, i.e., stimulated the growth of E. coli in SAPI+serum. Taken together, these data show that when low numbers of E. coli are inoculated into SAPI+serum, NE, DBS, and related catecholamines induce the enterobactin iron uptake system. This, in turn, facilitates iron sequestration from transferrin and indicates that the AI present in NE-conditioned SAPI+serum medium is enterobactin and its DBS breakdown products.

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Figures

FIG. 1.

FIG. 1.

Response of E. coli JPN10 to NE in SAPI (□) and in SAPI+serum (▪). The initial inoculum of JPN10 used for the assay assay was 144 ± 14 CFU/ml. Bacteria were incubated for 24 h at 37°C in a humid atmosphere containing 5% CO2, after which each sample was enumerated for viable counts. Each point represents the mean ± the standard deviation (SD) of triplicate samples. The dose-response curves are representative of three experiments performed on separate occasions. LOD, limit of detection.

FIG. 2.

FIG. 2.

Influence of iron on growth of E. coli JPN10 in SAPI, SAPI+serum (SS), and SAPI+serum with 50 μM NE (SSN). Bacteria were incubated for 24 h at 37°C in a humid atmosphere containing 5% CO2, after which each sample was enumerated for viable counts. The Fe3+ concentrations in the key are given in micromolar amounts. Each bar represents the mean ± the SD of triplicate readings. LOD, limit of detection.

FIG. 3.

FIG. 3.

Influence of NE on growth of E. coli strains with defects in enterobactin biosynthesis (entA [A]) and transport (fepA [B] and tonB [C]) in modified SAPI (SCTP) or SCTP+serum (SCTP+S), or SCTP+serum plus 50 μM NE (SCTP+SN). (A) JPN10, 13-6 (entA+ parent), and AN193 (entA mutant); (B) JPN10, AT2472 (fepA+ parent), and P1789 (fepA mutant); (C) JPN10, C600 (tonB+ parent), and GUC6 (tonB mutant). Each bar represents the mean ± the SD of triplicate samples. Bacteria were incubated for 24 h at 37°C in a humid atmosphere containing 5% CO2, after which each sample was enumerated for viable counts. The analysis of each mutant and parent strain was performed on at least two separate occasions. LOD, limit of detection.

FIG. 4.

FIG. 4.

Western blot showing the NE-dependent induction of the ferrienterobactin receptor protein, FepA. Outer membrane proteins were prepared from cultures grown in SAPI+serum (lane 2), SAPI+serum supplemented with 20 μM Fe3+ (lane 3), and SAPI+serum supplemented with 50 μM NE (lane 4). All outer membrane samples were prepared from cultures adjusted to an OD600 of 1.0 prior to the preparation of the outer membranes. After SDS-polyacrylamide gel electrophoresis, proteins were electrophoretically transferred to nitrocellulose and probed with a polyclonal antibody raised against FepA. The positions of the prestained molecular mass markers (Bio-Rad), with sizes given in kilodaltons, are shown in lane 1.

FIG. 5.

FIG. 5.

Growth response of E. coli JPN10 in SAPI+serum (SS) to DBS (A) and DBA (B). Bacteria were incubated for 24 h at 37°C in a humid atmosphere containing 5% CO2, after which each sample was enumerated for viable counts. Each point represents the mean ± the SD of triplicate samples. LOD, limit of detection.

FIG. 6.

FIG. 6.

Response of (A) fepA+ parent strain, AT2472, (B) fepA mutant strain P1798, (C) entA+ parent strain 13-6, and (D) entA mutant strain AN193 to DBS in modified SAPI+serum. Bacteria were incubated for 24 h at 37°C in a humid atmosphere containing 5% CO2, after which each sample was enumerated for viable counts. Each point represents the mean ± the SD of triplicate samples. LOD, limit of detection.

FIG. 7.

FIG. 7.

Influence of AI from strain 13-6 (entA+ parent) and AN193 (entA mutant) prepared from bacteria grown in iron-limited, mM9 on the growth of JPN10 in SAPI+serum. mM9 was modified as described in the Materials and Methods to support the growth of strain 13-6 and AN193. Uninoculated mM9 medium was used as a control. Spent culture supernatants from strain 13-6, AN193 and uninoculated mM9 medium were acidified and extracted with ethyl acetate, the solvent was removed, and the residue obtained was redissolved in mM9 medium to the original volume. Bacteria were incubated for 24 h at 37°C in a humid atmosphere containing 5% CO2, after which each sample was enumerated for viable counts. Each bar represents the mean ± the SD of triplicate samples. LOD, limit of detection.

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