The Neisseria meningitidis ZnuD zinc receptor contributes to interactions with epithelial cells and supports heme utilization when expressed in Escherichia coli

Abstract

Neisseria meningitidis employs redundant heme acquisition mechanisms, including TonB receptor-dependent and receptor-independent uptakes. The TonB-dependent zinc receptor ZnuD shares significant sequence similarity to HumA, a heme receptor of Moraxella catarrhalis, and contains conserved motifs found in many heme utilization proteins. We present data showing that, when expressed in Escherichia coli, ZnuD allowed heme capture on the cell surface and supported the heme-dependent growth of an E. coli hemA strain. Heme agarose captured ZnuD in enriched outer membrane fractions, and this binding was inhibited by excess free heme, supporting ZnuD's specific interaction with heme. However, no heme utilization defect was detected in the meningococcal znuD mutant, likely due to unknown redundant TonB-independent heme uptake mechanisms. Meningococcal replication within epithelial cells requires a functional TonB, and we found that both the znuD and tonB mutants were defective not only in survival within epithelial cells but also in adherence to and invasion of epithelial cells. Ectopic complementation rescued these phenotypes. Interestingly, while znuD expression was repressed by Zur with zinc as a cofactor, it also was induced by iron in a Zur-independent manner. A specific interaction of meningococcal Fur protein with the znuD promoter was demonstrated by electrophoretic mobility shift assay (EMSA). Thus, the meningococcal ZnuD receptor likely participates in both zinc and heme acquisition, is regulated by both Zur and Fur, and is important for meningococcal interaction with epithelial cells.

Fig 1

Homology at the C-terminal region between the N. meningitidis ZnuD protein and characterized heme receptors. The conserved motifs, the FRAP and NPNL boxes, and the highly conserved histidine residue are in boldface and marked by black lines and pound signs, respectively. The identical residues are indicated by asterisks. The numbers at either side of the sequences are the residue numbers. Nm, Neisseria meningitidis; Mc, Moraxella catarrhalis; Sd, Shigella dysenteriae; Ye, Yersinia enterocolitica; Pg, Porphyromonas gingivalis; Bp, Bordetella pertussis.

Fig 2

Interaction of ZnuD with heme. (A) Binding of heme to E. coli cells expressing various tagged ZnuD protein constructs under noninduced (gray bars) and induced (black bars) conditions. The binding of free heme to whole cells was calculated by the difference between the OD₄₀₀ of the cell-free supernatant and the control heme solution without cells (10 μM as 100%). ZnuD constructs were the following: SZ51, ZnuD-V5-His₆; 389, ZnuD-V5; and 390, ZnuD-Flag. The data are representative of three independent experiments performed in triplicate. The inset shows the expression level of ZnuD with or without arabinose induction as determined by anti-His Western blotting. Whole-cell lysates from equal numbers of bacteria based on the OD₅₅₀ readings were loaded. (B) ZnuD binding to heme agarose. Enriched outer membrane fractions prepared from E. coli expressing the ZnuD-V5-His₆ construct were used. Lane 1, total outer membrane proteins (12 μg) from noninduced cultures; 2, heme agarose-bound proteins from noninduced samples; 3, heme agarose-bound proteins from induced samples; 4, total outer membrane fraction from induced samples (12 μg). The location of ZnuD is indicated on the right, while the asterisks mark bands that are present only in the induced sample, possibly resulting from incomplete denatured aggregates and degraded proteins during SDS-PAGE sample preparations. The protein molecular size ladders are indicated on the left. (C) Free heme competition. Outer membrane proteins (15 μg) from induced samples were used, and the amounts of ZnuD bound to heme agarose were analyzed by anti-His Western blotting. Samples: 1, no treatment; 2, treatment with 0.02 N NaOH; 3 to 6, treatment with 0.2, 0.5, 1, and 5 μg/μl heme (Hm), respectively.

Fig 3

Heme-dependent growth assays. (A) E. coli IR1583 hemA strains carrying either pSZ51 or pBAD were resuspended in soft agar and then poured onto NBD plates with or without arabinose. Discs were placed, and 10-μl aliquots of PBS, 5 mM Fe⁺³, and 5 mM heme solution was applied. The data are representative of at least three independent growth assays. (B) N. meningitidis strains were resuspended from overnight plate cultures in soft agar and poured onto GC plates supplemented with 100 μM desferal. Aliquots (10 μl) of PBS, 25 mM Fe⁺³, and 5 mM heme solution then were applied to the discs.

Fig 4

Interaction of meningococci with epithelial cells. (A) Intracellular replication of IR4130 (WT), IR4130R (znuD), IR4130B (tonB), and IR4130RC (complemented [compl.] znuD)as determined by gentamicin protection assay after overnight infection at an MOI of 2. The gentamicin-resistant counts at 6 h after gentamicin treatment (t = 6 [T6]) were divided by the resistant counts immediately after gentamicin killing (t = 0 [T0]) and then normalized to that of the wild-type strain. Both the znuD and tonB mutants showed replication defects within cells compared to the wild type. The data represent the means and the standard deviations of two independent experiments. Statistically significant differences as determined by unpaired Student's t test with two-tailed hypotheses are marked with asterisks (∗, P < 0.05; ∗∗, P < 0.001). (B) Adherence and invasion efficiencies. The infection of A549 cells was initiated by low-speed centrifugation at an MOI of 100. The adherence abilities (black bars) relative to the initial bacterial input counts were examined after 2 h of infection. The invasion abilities (hatched bars) were determined immediately after gentamicin treatment, and the values were divided by the adhered bacterial counts. The ratios then were normalized to those of the wild-type strain. The data represent the means and the standard deviations from independent experiments (n ≥ 5). Statistically significant differences as determined by unpaired Student's t test with the two-tailed hypothesis are marked with asterisks (∗∗, P < 0.001).

Fig 5

Analysis of the znuD promoter. (A) Primer extension of znuD obtained with the PE1 primer. Lanes C, T, A, and G indicate the dideoxy sequencing reactions. The coding sequence is shown on the right, with an asterisk indicating the transcriptional start site. (B) The nucleotide sequence of the znuD promoter region cloned in the YT394 reporter strain (primers p-F1 and p-R1). The annotated ATG start codon and the transcriptional start site are boldfaced and underlined, while the putative −10 and −35 elements are boxed. The Zur binding site proposed by Stork et al. (55) is marked by a dashed line, while the putative Fur boxes are shown by three consecutive arrows. (C) Iron induction of znuD expression. Strains YT394 (WT, black bars) and PKT300 (zur mutant, gray bars) were grown in RPMI to mid-log phase, divided into aliquots, and then treated for 1 h with 10 μM iron, zinc, or heme as indicated. Cultures grown with no addition (NA) were used as the control. The values are reported as the averages and standard deviations of the β-galactosidase activity in Miller units from at least two independent experiments done in triplicate. Statistically significant differences were determined by unpaired Student's t test between the treated samples and the NA samples in the respective background and are marked with asterisks (∗, P < 0.001).

Fig 6

Interaction of the meningococcal Fur protein with the znuD promoter. (A) Dose-dependent binding of the (His)₆-Fur protein to the znuD promoter. The fur and dsbD promoters are included as a positive and a negative control, respectively. Lane 1 in each panel contains free probe, lanes 2 to 4 contain 0.2, 0.4, and 0.6 μg of Fur protein, respectively, for znuD and fur probes, and 0.4 and 1 μg of Fur protein was used in lanes 2 and 3, respectively, for the dsbD probe. (B) Competition EMSA. Lane 1, free probe; lane 2, probe with 1 μg of Fur protein; lanes 3 and 4, probe with 1 μg of Fur protein in the presence of 1 and 0.5 μg nonspecific DNA competitor (a PCR fragment of the misS coding sequence), respectively; lanes 5 and 6, probe with 1 μg of Fur protein in the presence of 1 and 0.5 μg specific DNA competitor, respectively.