Bacillus anthracis secretes proteins that mediate heme acquisition from hemoglobin

Abstract

Acquisition of iron is necessary for the replication of nearly all bacterial pathogens; however, iron of vertebrate hosts is mostly sequestered by heme and bound to hemoglobin within red blood cells. In Bacillus anthracis, the spore-forming agent of anthrax, the mechanisms of iron scavenging from hemoglobin are unknown. We report here that B. anthracis secretes IsdX1 and IsdX2, two NEAT domain proteins, to remove heme from hemoglobin, thereby retrieving iron for bacterial growth. Unlike other Gram-positive bacteria, which rely on cell wall anchored Isd proteins for heme scavenging, B. anthracis seems to have also evolved NEAT domain proteins in the extracellular milieu and in the bacterial envelope to provide for the passage of heme.

Figure 1. Bacillus anthracis isdX1 and isdX2.

(A) The B. anthracis isd locus contains eight open reading frames, including genes for sortase B (srtB), IsdC (a NEAT domain protein and sortase B substrate), IsdE1-IsdE2-IsdF (ABC membrane transporter), IsdG (heme mono-oxygenase), and two NEAT domain proteins of unknown function (IsdX1 and IsdX2). (B) Alignment of amino acid sequences of B. anthracis IsdX1 (Ba - BAS4443) with homologs from B. cereus (Bc - BC4548), B. thuringiensis (Bt - RBTH03454), and B. weihenstephanensis (Bw - KBAB44137). Arrow indicates the predicted signal peptide cleavage site. Black dots refer to amino acids that are not absolutely conserved. Amino acids 27–152 of IsdX1 represent a NEAT (near iron transporter) domain. Tyrosine residues 136 and 140, which are conserved in many NEAT proteins, are underlined.

Figure 2. B. anthracis secretes IsdX1.

(A and B) B. anthracis strains were grown in iron-replete or iron-depleted medium for 12 hours, followed by fractionation of cultures into secreted (S), cell wall (C), and lysate (L) fractions. Samples were analyzed by immunoblot with antibodies specific for IsdX1, L6 (ribosomal protein), SrtB (sortase B membrane protein), and IsdC (cell wall anchored protein). (C) Serum of guinea pigs that recovered from anthrax infections was used for immunoblot with purified GST-IsdX1, GST-IsdX2 or GST (left panel). The arrows denote SDS-PAGE mobilities of proteins (Coomassie, right panel) used for the immunoblot analysis. The migratory positions of molecular weight markers are indicated (kDa).

Figure 3. IsdX1 binds heme.

(A and C) Codons 27–152 of isdX1 and 30–859 of isdX2 were cloned into pGEX-2TK and the hybrid GST-fusions purified by affinity chromatography. Whole cells (WC), flow through (FT), wash and eluate fractions were analyzed on Coomassie-stained SDS-PAGE. The arrow in A identifies IsdX1, where GST had been removed with thrombin. The insets display tubes with eluate fractions (circled) containing red-brown pigment indicative of iron-porphyrin binding (red circles). (B and D) IsdX1 (20 µM) or IsdX2 (1 µM) were incubated with hemin and absorbance at 404 nm measured. Mean and standard deviation of three independent experiments were recorded.

Figure 4. IsdX1 removes heme from hemoglobin.

(A) Illustration of heme-transfer assay. Red stars indicate heme. (B, C) Glutathione-sepharose charged with GST-IsdX1 or GST (60 µM) was incubated with equimolar amounts of hemoglobin for 30 min, followed by centrifugation. Supernatant (B) and sediment (C) were assayed for absorbance at 404 nm. Insets display heme pigment in tubes with supernatant or sediment for GST-IsdX1 treated (red circles) or GST control (black circles). (D, E) Heme transfer was measured by adding increasing amounts of GST-IsdX1 or GST control (loaded on glutathione sepharose) to [⁵⁵Fe-heme]hemoglobin and radioactivity in sediment and supernatant samples recorded. Mean and standard deviation of three independent experiments are indicated.

Figure 5. Heme transfer from hemoglobin to IsdX1 or HasA.

HasA is a known hemophore in the Gram-negative pathogen Serratia marcescens. Glutathione-sepharose charged with GST-HasA, GST-IsdX1 or GST (200 µM) was incubated with increasing amounts of hemoglobin (50, 200, or 800 µM) for 30 min, followed by centrifugation. Sediment and supernatant samples were assayed for absorbance at 404 nm. Middle panel - Coomassie stained SDS-PAGE reveals GST proteins eluted from glutathione-sepharose. Lower panel - displays heme pigment in tubes with sediment derived from GST-HasA (red circles), GST-IsdX1 (red circles) or GST (black circles) treated hemoglobin samples. Mean and standard deviation of three independent experiments are recorded.

Figure 6. Association and dissociation of IsdX1 and hemoglobin.

Interactions between IsdX1 with hemoglobin (with bound heme) or apo-hemoglobin (lacking heme) were assessed by surface plasmon resonance (SPR).

Figure 7. Specificity of IsdX1 and IsdX2.

Heme acquisition when equimolar amounts of hemoglobin (Hb) or myoglobin (Mb) [800 µM] were incubated with GST-IsdX1 or GST-IsdX2 (60 µM). Mean and standard deviation of three independent experiments are recorded.

Figure 8. Transfer of heme from hemoglobin to NEAT domain proteins.

Purified GST hybrids with (1) S. aureus IsdC (S-IsdC), (2) B. anthracis IsdC (B-IsdC), (3) IsdX2, (4) IsdX1, or (5) GST control were incubated with hemoglobin and heme transfer measured as in Fig. 4. Mean and standard deviation of three independent experiments are recorded. Inset reveals the mobility of purified proteins on Coomassie stained SDS-PAGE.

Figure 9. B. anthracis requires isdX1 and isdX2 for scavenging of heme from hemoglobin.

B. anthracis strain Sterne (wild-type – WT) and isogenic ΔisdX1, ΔisdX2, or ΔisdX1/isdX2 variants were transformed with pLM5 vector, pisdX1 or pisdX2 (encoding IsdX1 or IsdX2 proteins, respectively). Bacteria were grown in iron-replete (LB) or iron-deplete medium (IDM) with the indicated concentrations of hemoglobin at 30°C for 16 hours. B. anthracis growth was assayed by plating 5 µL of a 1∶400 dilution of bacterial culture onto LB/ Km agar plates and colony forming units per mL (CFUs/mL) determined. Mean and standard deviation of three independent experiments are recorded.