Neutrophil gelatinase-associated lipocalin expresses antimicrobial activity by interfering with L-norepinephrine-mediated bacterial iron acquisition

Abstract

l-norepinephrine (NE) is a neuroendocrine catecholamine that supports bacterial growth by mobilizing iron from a primary source such as holotransferrin to increase its bioavailability for cellular uptake. Iron complexes of NE resemble those of bacterial siderophores that are scavenged by human neutrophil gelatinase-associated lipocalin (NGAL) as part of the innate immune defense. Here, we show that NGAL binds iron-complexed NE, indicating physiological relevance for both bacterial and human iron metabolism. The fluorescence titration of purified recombinant NGAL with the Fe(III).(NE)(3) iron complex revealed high affinity for this ligand, with a K(D) of 50.6 nM. In contrast, the binding protein FeuA of Bacillus subtilis, which is involved in the bacterial uptake of triscatecholate iron complexes, has a K(D) for Fe(III).(NE)(3) of 1.6 muM, indicating that NGAL is an efficient competitor. Furthermore, NGAL was shown to inhibit the NE-mediated growth of both E. coli and B. subtilis strains that either are capable or incapable of producing their native siderophores enterobactin and bacillibactin, respectively. These experiments suggest that iron-complexed NE directly serves as an iron source for bacterial uptake systems, and that NGAL can function as an antagonist of this iron acquisition process. Interestingly, a functional FeuABC uptake system was shown to be necessary for NE-mediated growth stimulation as well as its NGAL-dependent inhibition. This study demonstrates for the first time that human NGAL not only neutralizes pathogen-derived virulence factors but also can effectively scavenge an iron-chelate complex abundant in the host.

FIG. 1.

Chemical structures of neuroendocrine catecholamines shown in the order of their biosynthetic conversion (top), as well as siderophores with known affinity to NGAL (bottom). Enterobactin is produced by enteric bacteria, bacillibactin is produced by Bacillus spp., and parabactin is produced by Paracoccus denitrificans. Carboxymycobactins are produced by mycobacteria with species-dependent lengths of the carboxyalkyl side chain. In Mycobacterium tuberculosis, n = 1 to 5; in Mycobacterium avium, n = 2 to 8; in Mycobacterium smegmatis, n = 2 to 9.

FIG. 2.

SDS-PAGE of purified proteins used in this study. The recombinant proteins NGAL and Tlc, both fused at the C terminus with the Strep-tag II, as well as FeuA fused at the C terminus with the His₆ tag, all were produced in E. coli. M, protein size marker. At the bottom, the soaking of an apoNGAL crystal with Fe^III·(NE)₃ resulted in a deep red color, which indicates binding to the ligand pocket of the lipocalin.

FIG. 3.

Fluorescence titration of recombinant NGAL and FeuA with bacterial siderophores or neuroendocrine catecholamines. (A) One μM NGAL was titrated with the preformed Fe³⁺ complex of l-norepinephrine [Fe^III·(NE)₃]. (B) One μM NGAL was titrated with the preformed Fe³⁺ complex of l-epinephrine [Fe^III·(EPI)₃]. (C) NGAL (100 nM) was titrated with Fe^III·enterobactin (Fe^III·Ent). (D) NGAL (100 nM) was titrated with Fe^III·bacillibactin (Fe^III·BB). (E) FeuA (30 μM) was titrated with Fe^III·(NE)₃. (F) FeuA (100 nM) was titrated with Fe^III·BB. In all cases, Tyr/Trp fluorescence was excited at 280 nm and detected at 340 nm. Data were fit according to the law of mass action to determine the dissociation constants for protein-ligand complex formation (Table 1).

FIG. 4.

Cell growth assays with B. subtilis and E. coli wild-type (WT) and siderophore biosynthesis mutant strains. (A) Colony counts of B. subtilis WT (white bars) and E. coli WT (gray bars) cultures. (B to D) Colony counts of B. subtilis ΔdhbC (white bars) and E. coli ΔentC (gray bars) cultures. Strains were grown in iron-limited minimal medium with initial inocula of 10³ CFU/ml. Test supplements were made in different combinations and ligand concentrations (in μM) as indicated below the columns. After cultivation for 20 h, viable cells were counted. Bars represent average values of three independent biological experiments. Error bars indicate the corresponding standard deviations.

FIG. 5.

Determination of half-maximal inhibitory concentrations (IC₅₀) of NGAL with respect to bacterial growth in iron-limited cultures in the presence of 50 μM NE and 50 μM holoTf. (A) Dose-response curves of B. subtilis WT (filled circles) and ΔdhbC (filled squares) cultures. (B) Dose-response curves of E. coli WT (filled circles) and ΔentC (filled squares) cultures. Viable cell counts for each NGAL concentration were related to cell counts of control cultures in the absence of NGAL (corresponding to 100%). All data points represent average values of three independent biological replicates, and error bars indicate the corresponding standard deviations.

FIG. 6.

Cell growth assays with mutant strains of B. subtilis, ΔdhbC and ΔdhbC ΔfeuABC, defective in siderophore biosynthesis and siderophore biosynthesis as well as triscatecholate uptake, respectively. (A) Colony counts of B. subtilis ΔdhbC (white bars) and ΔdhbC ΔfeuABC (gray bars) cultures in the presence of NE. (B) Colony counts of B. subtilis ΔdhbC (white bars) and ΔdhbC ΔfeuABC (gray bars) cultures in the presence of EPI. Strains were grown in iron-limited minimal media with initial inocula of 10³ CFU/ml. Supplements were made to the cultures in micromolar concentrations as indicated below the columns. After cultivation for 20 h, viable cells were counted. Bars represent mean values of three independent biological replicates, and error bars indicate the corresponding standard deviations.

FIG. 7.

Structural comparison of the Fe^III·(NE)₃ complex with Fe^III·Ent. The coordinate set of Fe^III·Ent was taken from a partially refined crystal structure of its complex with NGAL (PDB entry 1IL6; courtesy of R. Strong) and energy minimized with CS Chem3D Pro 4.0 (left). Its interface with the ligand pocket of NGAL is indicated by the green line. The Fe^III·(NE)₃ complex was modeled starting from a similar geometry using the same software (right). Obviously, Fe^III·(NE)₃ would be able to form similar cation-π interactions, as they have been described for Fe^III·Ent bound to NGAL (25), even though the accommodation of the catechol side chains of NE might require some steric rearrangement at the opening of the ligand pocket. Illustrations were prepared with PyMOL (carbon and hydrogen, white; oxygen, red; nitrogen, blue; iron, orange; De Lano Scientific).