Hal Is a Bacillus anthracis Heme Acquisition Protein - Journal of

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Apr 23, 2012 - et al. annotated that the B. anthracis Sterne strain contained a gene ... Sterne 34F2 was extracted using a Wizard DNA purification kit (Promega,.
Hal Is a Bacillus anthracis Heme Acquisition Protein Miriam A. Balderas,a Christopher L. Nobles,a Erin S. Honsa,a Embriette R. Alicki,b and Anthony W. Maressoa Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA,a and Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas, USAb

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development.

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he extracellular concentration of free iron in mammals is kept very low (⬃10⫺18 M) (7, 9, 10, 43) as a means to prevent the formation of insoluble aggregates and metal-induced free radicals (51). Iron concentrations are regulated in the body by high-affinity iron-binding proteins such as transferrin and ferritin, which transport and store iron, respectively (1, 43, 51, 52). However, approximately 80% of the host iron pool is contained in heme (iron protoporphyrin IX) (8), which is mostly bound to hemoglobin, the major oxygen carrier protein in vertebrates (42, 47). There are two principal systems by which bacteria satisfy their need for iron. One mechanism is to secrete siderophores, small molecules that bind ferric iron through high-affinity interactions (43, 51). This mechanism is unlikely to account for iron import from heme sources owing to the strict sequestration of the iron in and the low off rates from heme and hemoproteins. In this regard, bacteria have evolved secreted or cell surface proteins that bind and acquire either free heme or heme from host hemoglobin (43, 51). The current paradigm is that imported iron is used as a cofactor for DNA synthesis and energy production, thereby promoting bacterial infection and replication in infected hosts (20, 51). Thus, the assimilation of iron from host heme is considered to be an important step in the maintenance and persistence of a bacterial infection. Bacillus anthracis, a Gram-positive spore-forming bacterium and the causative agent of anthrax, grows to high densities during infection of mammalian hosts, implying that this pathogen contains efficient and versatile iron-scavenging mechanisms. In 2004, Cendrowski et al. reported the identification of two siderophore systems in this pathogen, asb (anthracis siderophore biosynthesis) and bac (Bacillus anthracis catechol) (13). Functional loss of asb but not bac reduced the growth of B. anthracis in iron-depleted medium and virulence in an inhalational mouse model of anthrax disease. This defect was traced to an inability of the asb mutant spores to outgrow inside macrophages (13), leaving open the

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question of how bacillus attains needed iron when it reaches blood or host tissues. In 2006, Maresso et al. identified an eight-gene system with similarities to the then recently described isd (ironregulated surface determinant) locus, with one component, IsdC, being a heme-binding protein and important for bacillus growth on heme (34). However, the deletion of isdC did not attenuate virulence in guinea pigs, which suggested that Isd may be dispensable for anthrax disease progression (23). During a search for genes that are surface anchored by transpeptidase sortases, Gaspar et al. annotated that the B. anthracis Sterne strain contained a gene (bas0520) with an LGATG sorting signal (21). This gene is predicted to encode an N-terminal near-iron transporter (NEAT) domain, several internal leucine-rich repeat (LRR) regions, and a C-terminal sortase-like cell wall anchor (Fig. 1A). Analysis of this NEAT domain revealed sequence similarity to the NEAT domains of isdC (21%), isdX1 (29%), and bslK (49%) and the 5 NEAT domains of isdX2 (⬃29%) (this work). Furthermore, a recent study by Carlson at al identified the Ames homolog of bas0520 (gbaa0552) as being highly upregulated under conditions of iron starvation, and deletion of the gene resulted in an approximately 110-fold increase in the 50% lethal dose of B. anthracis Sterne 34F2 in an inhalation model of anthrax disease (11). Hypothesizing that BAS0520 is the missing factor needed for heme assimilation during anthrax disease, we demonstrate here that BAS0520 is a hemebinding protein that takes heme from mammalian hemoglobin and is necessary for growth on heme and hemoglobin in low-iron

Received 23 April 2012 Accepted 25 July 2012 Published ahead of print 3 August 2012 Address correspondence to Anthony W. Maresso, [email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.00685-12

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FIG 1 Functional annotation of Hal. (A) Hal was analyzed using a combination of PROSITE, SignalP, and KEGG. The arrowhead indicates the predicted site of signal peptidase cleavage. Hal is annotated as having a NEAT domain (residues 29 to 152, gray highlighted), 16 leucine-rich repeats (bold; labeled 1 through 16), and a C-terminal Gram-positive bacterium anchor (residues 1028 to 1070; underlined). (B) The NEAT domain of Hal was aligned with the eight NEAT domains of B. anthracis, the NEAT domain of IlsA, a protein involved in iron uptake in B. cereus, and Shr from Streptococcus pyogenes (14, 39). Hal contains two regions common to all NEAT domains, an N-terminal 310 helix of 4 to 6 amino acids and a C-terminal ␤ hairpin (bracketed regions). It is notable that, unlike heme-binding NEAT domains, Hal lacks a conserved tyrosine residue (instead, there is a phenylalanine) in the hairpin that is known to stabilize the interaction with the heme iron (hairpin, bold residues).

environments. With these new findings, we have appropriately assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520 and propose efforts to examine whether Hal is suited for vaccine or drug development. MATERIALS AND METHODS Bacterial strains. Escherichia coli strains (DH5 and BL21) were used for the cloning and amplification of hal and were grown in Luria broth (LB) supplemented with 50 ␮g/ml ampicillin (Fisher Scientific, Pittsburgh, PA). Wild-type B. anthracis strain Sterne (34F2) (46) was used for the growth studies and to generate a complete deletion in hal. For this purpose, hal was deleted by allelic replacement using the temperature-sensitive plasmid pLM4 (36). Briefly, genomic DNA of B. anthracis strain Sterne 34F2 was extracted using a Wizard DNA purification kit (Promega, Madison, WI), and 1,000 bp of the 5= and 3= sequences flanking the hal gene was PCR amplified with primer pairs hal-SmaI (5=-CCCGGGGCAA GGAAATGGTGACAGCAAAGTTGG-3=) and hal-KpnI (5=-GGTACCC

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GAGATATGATTACATTTAG-3=) as well as hal-KpnI (5=-GGTACCGG TGTAAATATATTAGAGAAGGAGG-3=) and hal-SacI (5=-GAGCTCCC CATTCATATGAAAATGATATTTCATATG-3=), respectively. Following ligation of the two fragments at the KpnI site, the 2-kb insert was cloned between the SmaI/SacI sites of pLM4 and plasmid DNA was amplified in the dam mutant E. coli strain K1077 prior to electroporation into B. anthracis. After transformation into strain Sterne, bacilli were first grown at 30°C (permissive temperature) on LB (20 ␮g/ml kanamycin) and then shifted to 43°C (restrictive temperature), followed by growth at 30°C to induce plasmid loss, thereby generating a B. anthracis ⌬hal strain. Bacteria were examined for kanamycin resistance by plating on LB agar, and DNA sequencing was performed to verify the presence or absence of wild-type and mutant allele nucleic acid sequences. Isogenic deletions in bslK and all eight genes of the isd system were also constructed using this procedure. Primer pairs 5= flank (bslK-SmaI [5=-CGTTTTGACGTTATCGTTTCAG3=] and bslK-KpnI [5=-GAGCGGAAAGCGTACTTATG-3=]) and 3= flank (bslK-KpnI [5=-GAGGCGCACAACATTCAC-3=] and bslK-SacI [5=-GTT

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TTTACATTACCGAAACCG-3=]) were used for bslK deletion. For the isd deletion, the primers were 5= flank (isd-SmaI [5=-GGTCTACTACTTGT GTATTTAG-3=] and isd-KpnI [5=-GCTAATTAAATAATGGGTAGAA G-3=]) and 3= flank (isd-KpnI [5=-CGCATGTACGACTAACCTTCC-3=] and isd-SacI [5=-CCATGACGATCGTCAATCCATG-3=]). To construct the complementation strain, the full-length hal gene was PCR amplified from the genomic DNA of B. anthracis strain Sterne 34F2 using forward (5=-GATCGATCGTCGACCATTTAGATTTATATATTTT GGAGG-3=) and reverse (5=-GATCGATCGGTACCCTAGTGGTGATG GTGATGATGCCTCTCCTCCTTCTCTAATA-3=) primer pairs with engineered SalI and SphI restriction enzyme sites, respectively. The PCR product was digested and cloned into a B. anthracis and E. coli shuttle vector, pUTE657 (27), such that expression was under the control of the isopropyl-␤-D-thiogalactopyranoside-inducible hyper-spac promoter, generating phal. Ligation products were transformed into E. coli DH5␣ and selected on 100-␮g/ml ampicillin agar plates. Plasmid DNA was then transformed into E. coli K1077 (with dam and dcm mutations) and then electroporated into B. anthracis ⌬hal strain to create B. anthracis ⌬hal phal. Protein purification. The NEAT domain (amino acids 29 to 152) of Hal (HalN) was purified as a glutathione S-transferase (GST) fusion protein similar to that described for other NEAT-domain proteins of B. anthracis (16, 19, 28, 34, 35, 48). Genomic DNA of B. anthracis strain Sterne 34F2 and primer pair halN-BamHI forward (5=-GATCGATCGGATCCG AGAATATGGCTGTACAAAGTC-3=) and halN-BamHI reverse (5=-GTA CGTACGGATCCTTACTACAAATGTACGCATATTCAC-3=) were used to amplify DNA encoding the NEAT domain. DNA was then cloned into the BamHI restriction site of pGEX2TK to create pgst-HalN, a vector with inducible expression of HalN as a GST fusion protein containing a thrombin cleavage site. pgst-HalN was then transformed into E. coli BL21 and grown in 100 ml of LB plus ampicillin at 37°C. Overnight cultures were then seeded into 1.5 liters of LB with ampicillin at 37°C and rotated at 250 rpm for 2 h, at which point isopropyl ␤-D-thiogalactopyranoside (final concentration, 1.5 mM; Sigma, St. Louis, MO) was added to the culture, which was incubated for an additional 2 h. Cells were next sedimented by centrifugation at 6,000 ⫻ g for 10 min and resuspended in phosphatebuffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4), and the cells were lysed by French press. The lysate was then centrifuged at 14,000 ⫻ g for 15 min, and the supernatant was sterilized with a 0.22-␮m-pore size cellulose filter. The filtrate was then subjected to affinity chromatography using 2 ml of glutathione-Sepharose resin (GE Healthcare, Piscataway, NJ) that was preequilibrated with 20 ml of PBS. Bound fusion protein GST-HalN was washed with 40 ml of PBS, GST-free HalN was released with the addition of 50 units of thrombin (Calbiochem, Darmstadt, Germany), and thrombin was removed from the HalN preparations using aminobenzamidine resin (Sigma). Prior to determining the binding properties of the NEAT domain, endogenous iron-porphyrin was removed by methyl ethyl ketone (MEK) treatment to generate apoprotein as described previously (49). The concentration of HalN was determined by the bicinchoninic acid assay (Pierce, Rockford, IL) or the Bradford assay (Bio-Rad, Hercules, CA). Typical yields using this purification method are approximately 4 mg/liter of E. coli culture. All protein preparations were stored at ⫺20°C. Heme binding to HalN. HalN (10 ␮M) was incubated with (0.25 to 5.0 ␮M) or without hemin chloride in PBS, pH 7.4, for 30 min at 25°C, and incubation was followed by a spectral analysis at from 250 to 650 nm using a DU800 spectrophotometer (Beckman-Coulter, London, United Kingdom). Heme acquisition from hemoglobin. GST-HalN (7.5 ␮M) was immobilized on 2 ml of glutathione-Sepharose resin, washed with 40 ml of PBS, and incubated with 1 ml of bovine methemoglobin (2.5 ␮M, Sigma) for 30 min at 25°C. Control columns consisted of a protein-only control (incubated with PBS) and another treated with hemoglobin with no protein immobilized. After 30 min, the columns were drained (hemoglobin

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fraction), washed 2 times with 10 ml of PBS, and treated with 0.5 ml of reduced glutathione (25 mM) for 5 min to elute bound GST-HalN. A wavelength scan from 250 to 650 nm was used to assess the relative amount of heme in each sample. Proteins samples were analyzed by SDSPAGE and silver stain (Bio-Rad silver kit). Association with hemoglobin. The interaction between HalN with methemoglobin was determined by surface plasmon resonance (SPR) using a BIAcore 3000 biosensor (Amersham Biosciences). Holo-bovine methemoglobin (in 50 mM Tris-HCl, pH 7.0) was covalently coupled to a CM5 sensor chip at 25°C to a density of 3,600 response units (RUs) using amine chemistry as previously described (30, 38). HalN (50 to 200 ␮M) in HBS-N (0.01 M HEPES, 0.15 M NaCl, pH 7.4) was injected at 20 ␮l/min for 300 s at 25°C. Binding data were attained using double referencing, where parallel injections of analyte are flowed over a CM5 surface that lacks hemoglobin (baseline) and these readings are subtracted from the experimental (HalN over hemoglobin) signal. Data were analyzed using BIAevaluation (version 4.1) software (Amersham Biosciences) after fitting the data to a 1:1 Langmuir binding model (15, 30, 38). Each concentration of HalN was injected twice, and each experiment was performed in triplicate. Growth studies. RPMI (Life Technologies, Grand Island, NY) was chelated of iron by incubation with 2.5 g of Chelex-100 per 100 ml of medium for 12 h at 4°C (cRPMI). B. anthracis Sterne 34F2 wild-type, ⌬hal, ⌬bslK, and ⌬isd (all containing empty plasmid pUTE657) strains and the complemented mutant (B. anthracis ⌬hal phal) were subcultured in LB overnight for 12 h at 37°C, and the bacterial pellets were washed with cRPMI and inoculated into cRPMI for 12 h at 37°C. Ten microliters of this culture at an optical density of 1.0 was then inoculated into 4 ml of fresh cRPMI with or without hemin (1, 10 ␮M) or hemoglobin (10, 100 ␮M), and growth (absorbance 600 nm) at 37°C was monitored from 2 to 16 h.

RESULTS

Hal annotation and NEAT-domain purification. The gene for hal is not localized to the isd locus and thus may code for a protein that is functionally separate from the Isd system. To gain insight into the function of hal, the amino acid sequence for this protein was analyzed using a combination of PROSITE, SignalP, and KEGG domain annotation programs (31, 40, 45) (Fig. 1A). The analysis revealed the presence of a putative N-terminal signal peptide (amino acids 1 to 21) and a proposed C-terminal Gram-positive bacterium anchor (GPA) region (amino acids 1028 to 1070). These two findings, along with the bioinformatic identification of Hal as a surface protein (the gene is designated BA0552 in the Ames strain of B. anthracis) (21), suggest that Hal is secreted through the general secretory pathway and tethered to the surface peptidoglycan, most likely by covalent attachment by a sortase transpeptidase. Furthermore, between the signal peptide and the GPA are 16 LRRs, with each repeat containing 21 amino acids. There are two clusters of these repeats; the first cluster contains 9 repeats and the second cluster contains 7, with each cluster separated by 171 amino acids. The exact function of bacterial LRRs is still unresolved, but several reports link them to protein-protein interactions (32, 33). The NEAT domain of Hal also contains two regions common to all NEAT domains, an N-terminal 310 helix of 4 to 6 amino acids and a C-terminal ␤ hairpin consisting of 6 to 8 residues (Fig. 1B, bracketed regions). However, unlike hemebinding NEAT domains, Hal lacks a conserved tyrosine residue (instead, it possesses a phenylalanine) in the hairpin that is known to stabilize the interaction with the heme iron (Fig. 1B, bold residue in hairpin region), thus drawing into question whether Hal is a functional heme-binding protein. Consistent with its observed expression under low-iron conditions (11), a putative 19-residue Fur box (12, 26) is located 112 bp upstream of the transcriptional

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TABLE 1 Fur box sequences of hal, bslK, and isda Gene

Fur box

Distance from ATG (bp)

Consensus hal bslK isd locus 1 isd locus 2 isd locus 3 isd locus 4 isdG locus 1 isdG locus 2

GATAATGATAATCATTATC GACAATGATAATGAAAATC GACATTGATAATCATTATC GAGAATGATAATCAATAAA GAGAATGATAATCAATAAT GAGAATAATTATCATTTTC TAAAATGAGAATAATTATC GATAATGATTTTCATTATC GACAATGATAATGATTTTC

⫺112 ⫺72 ⫺221 ⫺127 ⫺47 ⫺55 ⫺56 ⫺62

a The similarity of each putative Fur box for hal, bslK, and isd with a consensus Fur box sequence was determined. Deviations from the consensus sequence are shaded gray, and the distance of the box from the transcriptional start site is noted.

start site of hal. Since Fur is considered to be the primary repressor of genes involved in iron uptake, the presence of this Fur box before hal (and related genes) signifies regulation by iron availability (Table 1). Hal is a heme-binding protein. We reasoned that the most logical way to begin to deduce the function of Hal was to assess if it is involved in heme acquisition. As the first step in this process, we cloned the DNA encoding the putative NEAT domain of hal (HalN) into pGEX2TK to create a fusion to GST. Recombinant GST-HalN was expressed in E. coli and isolated using GST affinity chromatography. Following removal of the GST tag, pure HalN was attained and used for subsequent functional studies (Fig. 2A). To determine if the predicted HalN can associate with heme, the spectral properties of HalN were measured after purification from E. coli. Figure 2B (0 ␮M heme) shows the presence of a small peak of absorbance at 403 nm (Soret band), a diagnostic feature associated with the presence of iron-porphyrin in heme-binding proteins, as described previously (16, 19, 28, 34, 35, 48). This suggests that some HalN copurifies with heme during its purification from E. coli. To further test heme-binding function, the copurifying heme in the HalN preparation was removed with acid treatment, followed by methyl ethyl ketone treatment, and the apoprotein was titrated with hemin. As observed in Fig. 2B, the addition of hemin to HalN resulted in a clear absorbance increase and a red shift in the Soret band compared to reaction mixtures containing only hemin, which consistently showed a broad peak at 380 nm (compare the black to the gray bars). Further titration of hemin resulted in a gradual movement of the peak to the absorbance maximum of the control, an indication that HalN is saturated and consistent free hemin now dominates the absorbance spectrum. These data suggest that the NEAT domain of Hal is capable of binding heme, despite lacking a stabilizing tyrosine characteristic of all previously studied heme-binding NEAT domains. Hal can acquire heme from hemoglobin. Erythrocytes account for a large amount (85%) of the organismal heme (42), and hemoglobin is the most abundant iron reservoir (approximately 3 g out of the 4 g total host iron pool) (8, 50); thus, circulating hemoglobin may provide a source of heme for Hal during B. anthracis infection. To test this hypothesis, we coupled GST-HalN to glutathione-Sepharose and incubated the complex with or without bovine hemoglobin. After 30 min, GST-HalN and hemoglobin fractions were separated and heme content was assessed by Soret

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spectroscopy. As observed in Fig. 3A, GST-HalN incubated with hemoglobin yielded a higher Soret absorbance than GST-HalN incubated with a buffer control. Performing this experiment in triplicate and quantifying the ratio of the absorbance at 403 nm (heme) to that at 280 nm (GST-HalN) indicated that the difference in heme was approximately 3-fold between each group, suggestive of significant heme transfer to GST-HalN (Fig. 3B). As analyzed by SDS-PAGE and silver stain analysis, this effect was not due to hemoglobin contamination of the GST-HalN fraction (Fig. 3C; compare the migration of the eluted proteins to that of the hemoglobin standard). Taken together, these data indicate the NEAT domain of Hal is sufficient in the extraction of heme from hemoglobin. A Hal-hemoglobin interaction may facilitate heme removal. To determine if the observed heme transfer from hemoglobin to the NEAT domain of Hal is possibly mediated by a protein-protein interaction mechanism (active process), we assessed the potential binding of HalN to hemoglobin using surface plasmon resonance spectroscopy. Hemoglobin was coupled to a carboxymethyl surface, and increasing amounts of recombinant apo-HalN were infused over the surface. After fitting the data to a 1:1 binding model (30, 38), as observed in Fig. 3D, dose-dependent increases in the response units, a measure of physical association, were observed for chambers containing HalN and hemoglobin. These results suggest that the NEAT domain of Hal directly engages hemoglobin, a process that possibly mediates heme acquisition from hemoglobin. Loss of hal compromises growth on heme and hemoglobin. The analysis of the NEAT domain of Hal indicates that Hal is a heme-binding protein that actively acquires heme from host hemoglobin. To determine whether this protein plays a functional role in enhancing the growth of B. anthracis in low-iron, highheme environments, we generated an isogenic strain with a knockout in hal (⌬hal) and tested this strain for growth on heme and hemoglobin relative to that of a wild-type strain and two additional knockouts: one that harbors a complete deletion in all eight genes of the isd-like locus (⌬isd) and one that lacks the gene coding for bslK, the only other annotated NEAT protein in B. anthracis (⌬bslK). BslK is a surface protein that binds heme with a

FIG 2 Hal is a heme-binding protein. (A) HalN was expressed in E. coli as a GST fusion protein and purified by affinity chromatography. The SDS-polyacrylamide gel shows the fusion and GST-free (arrow) forms of HalN. (B) HalN (10 ␮M) from panel A was incubated with heme (1 to 10 ␮M; black lines), and the absorbance from 200 to 450 nm was recorded and compared to that for heme-only controls for all concentrations used (gray lines). About 10% of purified HalN copurified with bound heme (see the results for 0 ␮M), as determined using the pyridine hemochrome method (3). The data are representative of one of three independent determinations.

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Function of Hal

further studying Hal and uncovering its full function in B. anthracis growth and heme acquisition. In agreement with the biochemical results presented in Fig. 2 to 4, these data provide the first evidence that Hal functions as a bacillus hemoprotein that specifically acquires heme from host hemoglobin. DISCUSSION

FIG 3 The NEAT domain of Hal can acquire heme from methemoglobin. (A) GST-HalN (7.5 ␮M) was incubated with or without bovine methemoglobin (2.5 ␮M) for 30 min at 25°C, and proteins were separated by affinity chromatography using glutathione-Sepharose. Hb, hemoglobin. (B) Heme occupancy was calculated by taking the ratio of the Soret absorbance (heme, 403 nm) to the protein absorbance (280 nm) for three independent experiments. (C) Ten microliters of each elution was applied to an SDS-polyacrylamide gel, and GST-HalN or hemoglobin was detected by silver stain. Lane M, molecular mass markers; lane ST, standard representing an aliquot of methemoglobin to provide a reference for hemoglobin’s mobility upon SDS-PAGE. (D) Apo-HalN (50, 100, 200 nM) was infused over methemoglobin coupled to a carboxymethyl chip, and response units were measured for 300 s, followed by buffer infusion to monitor dissociation. Each concentration of HalN was injected twice, and each experiment was performed in triplicate. One representative experiment is shown.

very high affinity and whose exact role in B. anthracis iron homeostasis is not known. At low concentrations of heme (1.0 ␮M) and hemoglobin (10 ␮M), there were observable differences in the extent of growth between the wild-type and ⌬hal strain; however, these differences were not significant (Fig. 4C and D). When the concentrations of heme (10 ␮M) and hemoglobin (100 ␮M) were increased, significant differences in the growth of ⌬hal compared to that of wild-type B. anthracis were observed, with the ⌬hal strain growing poorly on both iron sources (Fig. 4A and B). Each strain grew equally well when grown in nutrient-rich LB (Fig. 4E), and the growth defect on heme or hemoglobin was partially or fully restored, respectively, by the plasmid expression of fulllength hal in the knockout strain. Together, these results suggest that the absence of hal leads to a growth defect of B. anthracis Sterne when heme or hemoglobin is the only iron source. Interestingly, the ⌬hal strain showed a higher growth defect than both the ⌬isd and ⌬bslK strains on heme or hemoglobin, a result that suggests that Hal may play a more prominent or unique role in iron uptake from heme iron than these other NEAT-containing proteins or systems. These data also highlight the importance of

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Here we report the first functional characterization of bas0520, hereby named hal (heme-acquisition leucine-rich repeat protein), a gene important for inhalational anthrax and upregulated under low-iron conditions (11). Our findings suggest that (i) the NEAT domain of Hal (HalN) can bind heme, despite lacking a stabilizing tyrosine, (ii) HalN can acquire heme from hemoglobin, (iii) a physical complex forms between HalN and methemoglobin, and (iv) Hal is important for growth on heme and hemoglobin, seemingly more so than Isd or BslK, which may explain the marked defect in virulence (in comparison to that of wild-type infection) observed for an inhalational anthrax murine model using a Sterne strain lacking this protein (11). It is now recognized that Gram-positive pathogenic bacteria utilize secreted or cell surface proteins containing NEAT domains to mediate the acquisition and import of heme (24, 25, 35, 37). B. anthracis contains five genes that harbor one or more NEAT domains (23, 28, 34, 35, 48). Three of these (isdX1, isdX2, and isdC) are part of the Isd locus, an array of eight genes proposed to encode a system that acquires heme from hemoglobin and delivers captured heme to the cell surface and across the cell membrane into the bacterial cytosol (19, 29, 34, 35). IsdX1 and IsdX2 are secreted proteins that extract heme from hemoglobin and deliver the heme to cell wall-bound IsdC (23, 29, 35). These are highly antigenic proteins, a finding that suggests an important role in heme-iron uptake during anthrax disease (22). Interestingly, a triple mutant lacking all three genes encoding the Isd proteins IsdX1, IsdX2, and IsdC was not reduced in virulence, as assessed by a guinea pig model of infection (23). The biochemical study of IsdX1 and IsdX2, however, has led to important insights into how bacillus NEAT-domain proteins mechanistically function, including the identification of NEAT-hemoglobin and NEAT-NEAT complexes (for transfer) and amino acids that facilitate heme and hemoglobin association (16, 29). Comparison of the properties of the NEAT domains of IsdX1 and IsdX2 to those of Hal yields interesting observations when considering the mechanism of heme coordination. For example, Hal has a phenylalanine in the fifth position of the heme-binding motif (YDKEF in Hal), a fiveresidue stretch of amino acids common to all proteins with NEATs (Fig. 1). For heme-binding NEAT domains, the first tyrosine in this region serves as the sixth axial ligand for the hemin iron (29). The second tyrosine hydrogen bonds to the first, an interaction that stabilizes the coordination (25, 41, 44). Both tyrosines are essential for heme stabilization in the NEAT domain, and all the NEAT-containing proteins from B. anthracis that contain the second tyrosine efficiently bind heme (25, 29, 41, 44). For example, the second NEAT domain of IsdX2 lacks the second tyrosine and consequently does not associate with heme (28, 29). We modeled the structure (Fig. 5) of the Hal NEAT domain using our published structure of IsdX1 (Protein Data Bank [PDB] accession number 3SZ6) as a template. The IsdX1 model demonstrated that tyrosine 112, conserved in all bacillus NEAT-containing proteins and required for heme association, is the sixth axial ligand (the iron-to-hydroxyl distance is 2.2 Å). Interestingly, phe-

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FIG 4 B. anthracis lacking hal grows poorly on heme and hemoglobin as the only iron source. Wild-type, ⌬hal, ⌬hal plus pUTE657hal (designated phal), ⌬bslK,

or ⌬isd B. anthracis strains were grown on hemin (10 ␮M, 1 ␮M) (A, C) or hemoglobin (100 ␮M, 10 ␮M) (B, D) in iron-chelated RPMI or LB (E) at 37°C, and growth was monitored by measuring the optical density at 600 nm (O.D.600) for 2 to 8, 10, 12, or 16 h. Each wild-type strain also contained the empty pUTE657 vector as a control.

nylalanine 116 is within 5 Å of this tyrosine, suggesting that the aromatic ring of F116 may function in a comparable way as a tyrosine aromatic ring, as found in other similar NEAT proteins (i.e., to stabilize the coordinating hydroxyl). Interestingly, there is a tyrosine on the opposite side of the heme-binding pocket in the 310 lip helix (ESYAT). Ekworomadu et al. observed this region in IsdX1 to regulate heme and hemoglobin binding (16), and others have proposed that the lip helix is important due to its location over the heme-binding pocket (41, 44). In Hal, the tyrosine residue in this region is in close proximity to the heme and extends above the distal side of the heme iron. A bistyrosine linkage, one on each side of the heme face, would be a novel finding for a bacterial protein with a NEAT domain. The binding of heme by Hal provides evidence that this rule is not strictly observed, meaning that the heme-iron association of this protein is likely different than that of other NEAT-containing proteins. The model also confirmed the presence of the lip-helix tyrosine directly over the heme iron. However, the distance between the lip-helix tyrosine hydroxyl and the heme iron is approximately 5 Å, seemingly too

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far to act as an iron ligand. Additional studies are required to determine the role of this tyrosine in heme binding and transfer; however, it is a possibility that the lip-helix tyrosine interacts with the heme porphyrin itself, strengthening the interaction between the heme and NEAT. A previous study by Tarlovsky et al. discovered and characterized the B. anthracis protein BslK as possessing a single NEAT domain that localizes noncovalently to the surface of B. anthracis, most likely via the three S-layer homology (SLH) regions (48). BslK was shown to bind heme with high affinity and transfers heme to IsdC. The role of this protein in B. anthracis pathogenesis or heme uptake has not yet been tested. However, it is clear that deletion of bslK does not render B. anthracis unable to grow on heme or hemoglobin (Fig. 4). Interestingly, the ⌬bslK strain grew better than the isogenic wild-type strain on hemoglobin (and heme at later time points; Fig. 4). This may be partially explained by considering the high affinity of BslK for heme (almost no heme dissociation is observed in a 24-h period) (48). In the absence of bslK, Hal and Isd systems have access to a larger heme pool, thus

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Function of Hal

FIG 5 Structural model of the NEAT domain of Hal. The structural model of the NEAT domain of Hal was created using IsdX1 as the homology template in the SWISS-MODEL workspace (2, 8). The resulting PDB file was then manipulated in PyMOL (PyMOL Molecular Graphics System, version 1.5.0.1; Schrödinger, LLC). The 310 helix residues (EESYAT) are shown in cyan, and the ␤ hairpin containing the heme-binding sequence (YDKEFKIQ) is demonstrated in yellow. Hemin porphyrin (magenta) was modeled into the NEAT domain heme-binding pocket using Coot (2, 6, 17). Oxygen atoms are red, and nitrogen atoms are dark blue. (B) The proximal and distal sides of the heme are shown, demonstrating the interaction of heme with nearby residues of Hal.

possibly allowing these two systems to function more efficiently. This hypothesis is currently being tested. Gaspar et al. searched the sequenced genome of B. anthracis for putative sortase-anchored proteins and described bas0520 as coding for a protein with a proposed GPA (21). In fact, Hal contains two of the three features that are common for cell wall-attached proteins, including a hydrophobic and polybasic stretch of residues at the C terminus. However, a conserved putative proline observed in the second position of the canonical sortase A pentapeptide recognition motif (LPXTG) is missing in the sequence for Hal (the closest resemblance is LGATG). More recently, Carlson et al. noted that hal is upregulated under low-iron conditions and found the Sterne strain lacking this gene to demonstrate an approximately 100-fold reduction in virulence in a mouse model of inhalational anthrax (11). The putative GPA motif, the proposed importance of hal in virulence, the presence of a heme-binding NEAT domain, and the above-mentioned results all suggest that Hal may be an important mediator of iron uptake from blood sources. Indeed, findings in this report include data that support this hypothesis and possibly provide a link between anthrax disease progression and heme assimilation by this protein from hemoglobin. In addition to a NEAT domain, Hal contains 16 LLR sequences. The presence of evenly spaced repeating units of leucine allows a protein or domain to fold into a horseshoe shape with the hydrophilic residues exposed to solvent (32, 33). The internal leucines create a hydrophobic core that may enhance interactions with proteins. LRRs are found in evolutionarily diverse proteins harboring various numbers of copies of this structural motif ranging from 1 to 30 (32, 33). Functional attributes of LRRs include the

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modulation of cell adhesion and signal transduction, especially in eukaryotic cells. However, bacterial LRRs have not been extensively studied. A well-noted Gram-positive bacterial LRR protein is Listeria monocytogenes internalin (InlA), a surface protein that binds the host receptor E cadherin, a process that mediates the invasion of this pathogen into epithelial cells, which suggests the importance of the LRR region in the invasion process (4). The Streptococcus pyogenes protein Slr, which also contains several LRRs, binds to type 1 collagen, thereby showing that LRRs participate in bacterial adhesion to tissue components (5). LRR regions are also associated with bacterial toxins or effectors (e.g., the type III secreted protein YopM from Yersinia pestis) (18). These studies suggest that at least some fraction of LRR-containing proteins in bacteria promote the interaction of bacteria with host tissues, a process that likely contributes to pathogenesis. Interestingly, although LRRs may function to drive protein-protein interactions, work here suggests that Hal’s NEAT domain alone is sufficient for the interaction with hemoglobin. This interaction may drive heme transfer, although proof of this would require knowledge of the rates of heme loss from hemoglobin and transfer to Hal. We also cannot rule out the possibility that the LRRs strengthen this association or allow binding to other serum hemoproteins, such as the hemoglobin-haptoglobin complex, hemopexin, or other ironbinding proteins (14, 41). Indeed, the IslA protein from Bacillus cereus, which contains a NEAT domain and several LRRs, binds heme, hemoglobin, and ferritin (14). We are currently evaluating the role of the LRRs in the heme acquisition functions of Hal. One question raised by this study is why B. anthracis would require three heme uptake systems that evolved separately (Isd, BslK, and Hal). There are two possible reasons for this when considering an infection with B. anthracis. First, each system may be differentially expressed under certain conditions, in certain tissues, or at different times and thus plays a functional role at a different point during the infection. For example, deletion of the biosynthetic operon that encodes the siderophore anthrachelin (petrobactin) in B. anthracis results in a loss of spore germination in macrophages (13). A second possibility is that bacillus has evolved multiple systems to increase the overall rate of heme acquisition. In this regard, each component is functionally redundant, but collectively, the presence of multiple systems ensures that the cell maximizes its heme uptake capabilities. However, we cannot rule out the possibility that there are additional unrecognized functions for these proteins that impact our results, especially when one considers that Hal and BslK both contain uncharacterized protein domains (LRRs and SLH, respectively). It is clear from the growth studies whose results are presented in Fig. 4 that the uptake of heme in this pathogen may be a complex process with inputs from multiple systems. For example, in the absence of isd, bacilli seem to grow better than the wild-type strain on high concentrations of heme or hemoglobin. The simplest explanation is that in the absence of one system (BslK or Isd) at these concentrations of heme or hemoglobin, less overall heme is transported into the cell, leading to less heme toxicity and subsequently greater growth. Conversely, in the absence of a system, the cells may be sensing that they are iron starved and thus increase the expression of hal, which enhances growth. We are currently devising experiments to differentiate between these possibilities. The results presented here allow us to suggest a basic model for the function of Hal (Fig. 6). Upon entry into a mammalian host, B. anthracis encounters a low-iron environment and Fur-mediated

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FIG 6 Model for the function of Hal. Upon exposure of B. anthracis to a low-iron environment, Hal is expressed and covalently coupled by a sortase to the cell wall via its C-terminal GPA. Surface Hal may have its N-terminal NEAT domain exposed to the extracellular environment, positioned to interact with released hemoglobin. Once bound, Hal extracts the heme from hemoglobin and retains the heme through a unique heme-protein linkage, eventually transferring the bound heme to a downstream membrane-localized transporter. The LRRs may aid in this process or provide for bacillus binding to other iron-containing or host tissue proteins. Either the heme or the liberated iron is then used in essential biological processes, which enhances replication in iron-limiting environments. CW, cell wall; M, cell membrane.

repression of the gene is relieved. As Hal is passed through the Sec secretion system, the N terminus is recognized by one of the three B. anthracis sortases, possibly sortase A, thereby covalently attaching the protein to the cell wall. Upon exposure to blood or tissues, the NEAT domain temporarily associates with released hemoglobin, and the heme is removed and passed to an unidentified transporter(s) associated with the cell membrane. The LRRs may aid this protein-protein interaction or, alternatively, initiate binding to additional host proteins. The import of heme promotes bacterial replication and, eventually, anthrax disease, presumably through the use of the iron in key cellular processes. ACKNOWLEDGMENTS We thank Preeti Zanwar for a critical reading of the manuscript. This work was supported by a grant (AI069697 to A.W.M.) from the National Institutes of Health.

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