Analysis of haptoglobin and hemoglobin-haptoglobin interactions with the Neisseria meningitidis TonB-dependent receptor HpuAB by flow cytometry

Abstract

Neisseria meningitidis expresses a two-component TonB-dependent receptor, HpuAB, which mediates heme-iron (Hm-Fe) acquisition from hemoglobin and hemoglobin-haptoglobin complexes. Due to genetic polymorphisms in the human haptoglobin gene, haptoglobin (and hemoglobin-haptoglobin) exists as three structurally distinct phenotypes. In this study, we examined the influence of the haptoglobin phenotype on the interactions of HpuAB with apo-haptoglobin and hemoglobin-haptoglobin. Growth assays confirmed that HpuAB utilizes hemoglobin-haptoglobin more efficiently than hemoglobin as an Fe source and revealed a preference for human-specific, polymeric 2-2 or 2-1 hemoglobin-haptoglobin complexes. We developed a flow cytometry-based assay to measure the binding kinetics of fluorescein-labeled ligands to HpuAB on live, intact meningococci. The binding affinity of HpuAB for hemoglobin-haptoglobin phenotypes correlated well with the ability of each ligand to support Neisseria meningitidis growth, with higher affinities exhibited for types 2-2 and 2-1 hemoglobin-haptoglobin. Saturable binding of Hb and apo-haptoglobin suggested that HpuAB-mediated utilization of hemoglobin-haptoglobin involves specific interactions with both components. In contrast to previous studies, we detected binding of HpuB expressed alone to hemoglobin, apo-haptoglobin, and hemoglobin-haptoglobin of all three phenotypes. However, in the absence of HpuA, the binding capacity and/or affinity of the receptor was reduced and the dissociation of hemoglobin was impaired. We did not detect binding of HpuA alone to hemoglobin, apo-haptoglobin, or hemoglobin-haptoglobin; however, the lipoprotein is crucial for optimal recognition and use of ligands by the receptor. Finally, this study confirmed the integral role of TonB and the proton motive force in the binding and dissociation of Hb and hemoglobin-haptoglobin from HpuAB.

FIG. 1.

Growth kinetics of HpuA⁺B⁺ DNM140 with Hb and HbHp complexes. Meningococci expressing wild-type HpuAB were grown overnight on Fe-depleted CDM0 plates before being used to inoculate CDM0 broth cultures supplemented with Hb, type 1-1 HbHp, type 2-2 HbHp, or type 2-1 HbHp. Control cultures grown in Fe-replete (CDM100) and Fe-depleted (CDM0) media were included. The data presented are the averaged results of two independent experiments.

FIG. 2.

(A) Binding kinetics of Hb. Binding of fluorescein-labeled Hb to N. meningitidis strains DNM140 (HpuA⁺B⁺), DNM68 (HpuA⁻B⁻), DNM69 (HpuA⁻B⁺), and DNM143 (HpuA⁺B⁻) was detected using an Alexa Fluor 488 anti-fluorescein conjugate and measured by flow cytometry. The geometric mean fluorescence (AU) is shown as a function of fluorescent-ligand concentration. Binding at each ligand concentration was assessed in duplicate, and the data points shown represent the mean of at least two independent experiments. Error bars indicate the standard error of the mean for each combined data set. (B) Hb binding data for DNM68, DNM69, and DNM143 from panel A were plotted on a smaller y-axis range to illustrate the low-capacity binding to DNM69 for comparison with DNM140 (A). Error bars for DNM68 and DNM143 are too small to be seen due to the scale of the graph.

FIG. 3.

(A) Kinetics of binding of types 1-1, 2-2, and 2-1 HbHp to strain DNM140 (HpuA⁺B⁺). The data are presented as described in the legend to Fig. 2. The data for each ligand represent the mean of at least three independent experiments conducted in duplicate (total of six data sets per data point on the graph). (B) Kinetics of binding of types 1-1, 2-2, and 2-1 apo-Hp to strain DNM140. As above, data presented are the mean of three separate assays in which each sample was assayed in duplicate. (C) Binding of all three apo-Hp and HbHp phenotypes to DNM68 (HpuA⁻B⁻), DNM69 (HpuA⁻B⁺), and DNM143 (HpuA⁺B⁻). The height of each bar represents the amount bound on incubation of cells with 1 μM each fluorescein-labeled ligand. (D) Ability of Hb and apo-Hp to compete with HbHp for binding to strain DNM140. Cells were mixed with 150 nM FEX-labeled type 1-1 HbHp and 1.5 nM to 7.5 μM unlabeled competitors (type 1-1 HbHp, type 1-1 apo-Hp, and Hb). Results are expressed as the percentage of total binding, with 100% binding determined from control samples to which no competitor was added. Each data point represents the mean of two independent assays, with each sample assayed in duplicate.

FIG. 4.

Hb and HbHp binding to deenergized HpuAB. The data are presented as described in the legend to Fig. 2. Each data point represents the mean of two independent experiments conducted in duplicate. (A) Isotherm of Hb binding to wild-type (DNM140) and deenergized (DNM146) HpuAB. (B) Flow cytometry histogram depicting FEX-Hb binding to TonB⁺ DNM140. From left to right, peaks correspond to unstained control (grey) and 20, 50, 100, 200, 500, and 1,000 nM Hb. (C) Flow cytometry histogram of FEX-Hb binding to TonB⁻ DNM146. The shaded grey peak is the fluorescence of the unstained negative control. Ligand was added at same concentrations as used for the experiment panel B. (D) Isotherm of type 1-1 HbHp binding to wild-type and deenergized HpuAB. (E) Histogram of flow cytometry analysis of HbHp binding to DNM140. Left to right, fluorescence peaks of unstained control (grey) and 10, 25, 50, 100, 250, and 500 nM ligand. Note that the 250 and 500 nM (bold) peaks are superimposed. (F) Histogram of type 1-1 HbHp binding to DNM146. Ligand was added at same concentrations as used for the experiment in panel E. Fluorescence peaks of the 100, 250, and 500 nM (bold) samples are superimposed.

FIG. 5.

Energy-dependent dissociation of Hb and HbHp from HpuAB. DNM140, DNM146, and CCCP-treated DNM140 cells were equilibrated with 200 nM FEX-labeled Hb (A) or type 1-1 HbHp (B). Then, 20-fold excess unlabeled competitor was added at various time points and the amount of labeled ligand remaining bound to cells over time was determined. Control samples to which no competitor was added defined 100% binding. Each strain was tested twice in duplicate for each ligand (total of four data sets).