Ferric enterobactin binding and utilization by Neisseria gonorrhoeae

Abstract

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 microM, but growth stimulation was abolished when an omega (Omega) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 microM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Omega insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.

FIG. 1

FetA cross-reactivity with FepA MAbs. Western blots were probed with FepA MAb 2 (A) or FepA MAb 35 (B). Whole-cell lysates were prepared from iron-stressed bacteria. Lanes: 1, E. coli BN1071; 2, FA1090 (wild type); 3, FA6959 (fetA::Ω).

FIG. 2

FetA conservation of the FepA binding site. This lineup shows sequence conservation between FepA and FetA in the central region of the FepA structure bounded by residues 255 to 336. Residues within this region are implicated in ferric enterobactin binding (8a, 28). Shading indicates similar amino acids, and outlining indicates amino acid identities. Amino acids indicated in bold lettering are important in ferric enterobactin binding of FepA; the corresponding residues in FetA are also in bold. The lines above FepA residues 269 to 281 and residues 317 to 336 indicate extracellular loops L3 and L4, respectively, as determined by the recently solved crystal structure of FepA (8).

FIG. 3

FetB expression. A Western blot was probed with FetB-peptide polyclonal antiserum. Whole-cell lysates were prepared from iron-stressed bacteria. Lanes: 1, FA1090 (wild type); 2, FA7015 (fetB::Ω); 3, FA6959 (fetA::Ω).

FIG. 4

Growth stimulation by ferric enterobactin. ○, FA1090 with no added ferric enterobactin; solid symbols, cultures containing 10 μM enterobactin. ●, FA1090 (wild type); ■, FA6959 (fetA::Ω); ⧫, FA6990 (tonB::cat); ▴, FA7015 (fetB::Ω). The graph is representative of a minimum of three experiments.

FIG. 5

FetA expression in gonococcal strains. Whole-cell lysates were prepared from iron-stressed cultures of FA1090 (wild type) (lane 1), FA6959 (fetA::Ω) (lane 2), FA6990 (tonB::cat) (lane 3), FA6991 (fetA::Ω tonB::cat) (lane 4), and FA7015 (fetB::Ω) (lane 5). The Western blot was probed with FetA polyclonal antiserum.

FIG. 6

Ferric enterobactin binding by gonococci. The amount of [⁵⁹Fe]enterobactin bound (in picomoles per 10⁹ cells) was determined as a function of ferric siderophore concentration. (A) Ferric enterobactin (FeEnt) binding to wild-type (FA1090) (●) and fetA mutant (FA6959) (○) strains of N. gonorrhoeae. (B) FetA-specific binding (see the text) of ferric enterobactin by FA1090 (wild type) (●), FA6990 (tonB) (■), and FA7015 (fetB) (▴). Binding to wild-type E. coli K-12 strain BN1071, which expresses chromosomally encoded FepA, is also shown (⧫). The K_d values for binding to FA1090, FA6990, FA7015, and BN1071 were 5.7, 4.17, 6.3, and 0.018 μM, respectively; the mean standard error on these measurements was 30.4%. The capacities of the four strains were 31, 34, 22, and 32 pmol/10⁹ cells, respectively; the mean standard error on these measurements was 11.7%. For FA1090 and FA6990, data were collected from six or more experiments and the mean values are plotted. For FA7015, the data represent the mean of three separate experiments.