Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization

Abstract

The pathogenesis of human infections caused by the gram-positive microbe Staphylococcus aureus has been previously shown to be reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing three cell wall-anchored proteins (IsdA, IsdB, and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Here we report that S. aureus can utilize the host hemoproteins hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. We demonstrate that staphylococci capture hemoglobin on the bacterial surface via IsdB and that inactivation of isdB, but not isdA or isdH, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of S. aureus to utilize hemoglobin as an iron source. Stable-isotope-tracking experiments revealed removal of heme iron from hemoglobin and transport of this compound into staphylococci. Importantly, mutants lacking isdB, but not isdH, display a reduction in virulence in a murine model of abscess formation. Thus, IsdB-mediated scavenging of iron from hemoglobin represents an important virulence strategy for S. aureus replication in host tissues and for the establishment of persistent staphylococcal infections.

FIG. 1.

Growth of S. aureus using hemoproteins as a sole iron source. S. aureus strains were grown in iron-free NRPMI+ supplemented with 0.5 μM hemoglobin (A), 2 μM myoglobin (B), or 2 μM hemopexin (C). Bacterial growth was determined by measuring the OD₆₀₀ of cultures. Solid black lines represent S. aureus wild-type strain Newman, whereas dashed black lines indicate growth in the absence of hemoproteins. Data represent the mean ± the standard deviation of triplicate experiments.

FIG. 2.

Growth of S. aureus using intracellular hemoglobin as a sole iron source. (A) Immunoblot assay of extracts from K-562, an erythrocyte precursor cell line that was left uninduced (U) or induced (I) by using an anti-hemoglobin antibody as a measurement of hemoglobin expression. (B) S. aureus strain Newman was cultured in iron-free medium in the presence of K-562 cells left uninduced (black line) or induced (gray line) for the expression of hemoglobin (Hb). Data represent the mean ± the standard deviation of triplicate bacterial enumerations on agar plates. Asterisks denote statistically significant differences from the wild type as determined by Student's t test (P < 0.05).

FIG. 3.

Hemoprotein binding to S. aureus surface. Shown are results of FACS-based assays measuring the binding of hemoglobin (Hb; A), myoglobin (Mb; B), and hemopexin (Hp; C) to the surface of different strains of S. aureus. MFI, mean fluorescence intensity. Results represent the mean ± the standard deviation from triplicate determinations. Asterisks denote statistically significant differences from the wild type (WT) as determined by Student's t test (P < 0.05).

FIG. 4.

Hemoglobin binding to whole S. aureus cells. (A) Whole cells of the indicated S. aureus strains were incubated with various concentrations of hemoglobin (Hb; micrograms per milliliter), followed by immunoblotting with an antiserum specific for Hb. The left side represents hemoglobin, and the right side represents the loading control. (B) Whole cells of the complemented isdB mutant strain (ΔisdB/pOS1IsdB) and control strains containing the pOS1 plasmid without the isdB gene (i.e., wild type [WT]/pOS1 and ΔisdB/pOS1) were grown in iron-depleted (−Fe) or iron-replete (+Fe) medium. Whole cells were then incubated with various concentrations of hemoglobin (micrograms per milliliter), followed by immunoblotting with an antiserum specific for hemoglobin. On the left is hemoglobin, and on the right is the loading control. (C) S. aureus Newman (wild type) and the isogenic ΔisdB and Δfur mutant strains were grown in iron-depleted (−Fe) or iron-replete (+Fe) medium. Whole cells were then analyzed by immunoblotting with an antiserum specific for IsdB.

FIG. 5.

Growth of isdB mutants using hemin or hemoglobin as the sole iron source. (A) S. aureus strains were grown in iron-free medium continuously supplemented with hemin (2 μM) or hemoglobin (0.5 μM) as an iron source or without iron (−Fe). (B) S. aureus strains were grown in iron-free medium preincubated with hemin (2 μM) or hemoglobin (0.5 μM) for 30 min or not. Cells were then washed and cultured in iron-free NRPMI+. Bacterial growth was determined by measuring the OD₆₀₀ of cultures at 6, 9, and 12 h. Results represent the mean ± the standard deviation from triplicate determinations. Asterisks denote statistically significant differences from the wild type (WT) as determined by Student's t test (P < 0.007).

FIG. 6.

Contribution of IsdB-mediated hemoglobin binding to staphylococcal pathogenesis. S. aureus colonization of murine spleen (A) or kidney (B) tissue was measured by tissue homogenization, dilution, and colony formation on agar medium. The horizontal gray line represents the mean log CFU on the y axis. The horizontal black line represents the limit of detection. Each data point represents the number of bacteria (CFU) per milliliter of tissue homogenate in a single animal. Asterisks denote statistically significant differences between the wild-type (WT) and mutant strains as determined by Student's t test (P < 0.03).