Bacillus anthracis IsdG, a Heme-Degrading Monooxygenase
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《细菌学杂志》
Department of Microbiology, University of Chicago, Chicago, Illinois 60637
ABSTRACT
Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling the heme binding of known heme oxygenases. Formation of IsdG:hemin complexes in the presence of a suitable electron donor, e.g., ascorbate or cytochrome P450 reductase, promotes catalytic degradation of hemin to biliverdin with concomitant release of iron. IsdG is required for B. anthracis utilization of hemin as a sole iron source, and it is also necessary for bacterial protection against heme-mediated toxicity. These data suggest that IsdG functions as a heme-degrading monooxygenase in B. anthracis.
INTRODUCTION
The ability to acquire iron during infection is an essential attribute for many bacterial pathogens of vertebrates (4). This is due to the requirement for iron in numerous cellular processes that are fundamental for life, such as DNA replication, energy generation, and protection against reactive oxygen species (4). A primary human defense mechanism against bacterial infection is to severely limit available iron through the production of iron binding proteins. The most abundant group of iron binding proteins in humans are hemoproteins, polypeptides that utilize heme as a cofactor (12). Heme, a tetrapyrrole encircling an individual iron atom within the macrocyclic conjunction of the heme porphyrin ring, and hemoproteins represent approximately 80% of the iron in humans or animals (12). To successfully mediate infection, most bacterial pathogens have developed systems capable of extracting heme from human hemoproteins and subsequently utilizing this cofactor as both a heme and iron source (4, 7, 20, 45, 48). The molecular mechanisms whereby gram-negative bacterial pathogens acquire iron from heme are well characterized (4, 7, 20, 48). In contrast, the molecular mechanisms involved in heme iron scavenging in gram-positive pathogens are only now beginning to emerge (3, 28, 30, 45, 54). Nevertheless, heme has been shown to be the preferred source of iron during the pathogenesis of the gram-positive pathogen Staphylococcus aureus (44).
Bacillus anthracis is a gram-positive spore-forming rod and the causative agent of the disease anthrax (27). The systemic form of anthrax begins upon engulfment of the bacterial spores by resident macrophages, followed by intracellular germination and subsequent replication and amplification of the vegetative form of bacilli (13, 22, 23). Ultimately, vegetative cells of B. anthracis escape macrophages and enter circulation where they multiply in the bloodstream of infected individuals, at times reaching cell densities as high as 109 per ml (29). This large amount of growth necessitates a rapid accumulation of nutrients for the organism to satisfy its nutritional needs during replication. The only nutrient thought to be limiting for growth of bacterial pathogens during infection is iron (8), suggesting that efficient mechanisms for iron acquisition exist in B. anthracis. This hypothesis is corroborated by the presence of 15 putative ATP-binding cassette-type uptake systems specific for iron and two gene clusters exhibiting homologies consistent with siderophore synthesis encoded in the genome of B. anthracis (36). The production of two separate siderophores has been experimentally confirmed in this bacterium (9, 19), one of which is required for virulence in a mouse model of systemic infection (9). In addition to these siderophores, B. anthracis is capable of using heme, hemoproteins, iron-transferrin, and certain heterologous siderophores (xenosiderophores) as iron sources for growth (19). The molecular mechanisms responsible for the utilization of these iron sources are as yet unknown.
For bacterial pathogens to utilize heme as a nutrient source, the heme-bound iron atom must be liberated through opening of the porphyrin ring. Structural comparisons among known heme oxygenases identified two enzyme families capable of degrading heme to biliverdin and liberating iron in the presence of a suitable electron donor. The first is catalyzed by members of the heme oxygenase family, a ubiquitous enzyme family whose members have been identified in all kingdoms, including the bacterial pathogens Neisseria gonorrhoeae (59), Neisseria meningitidis (59), Pseudomonas aeruginosa (35), Corynebacterium diphtheria, and Corynebacterium ulcerans (38). Heme oxygenase family members are characterized by a conserved histidine residue in the N-terminal portion of the protein and a GXXXG motif required for catalytic activity (53).
Crystal structure analyses revealed that heme oxygenase family members function as monomeric alpha helical proteins with a single active site (25, 39, 40, 47, 53). The second family of heme-degrading monooxygenases, the IsdG family, is found exclusively in gram-positive bacteria (43, 45, 55). The genome of S. aureus encodes two IsdG family members, IsdG and IsdI, which are part of the iron-regulated surface determinant (isd) system of heme uptake (30). These enzymes are responsible for the degradation of hemin to free iron and biliverdin in the presence of an electron donor in vitro and confer the ability to utilize hemin as a sole iron source on a heme oxygenase mutant of C. ulcerans (43). The crystal structures of S. aureus IsdG and IsdI demonstrate that these enzymes fold in a dimeric beta-barrel type configuration encompassing two separate active sites for heme degradation (55). Although the crystal structure of an IsdG family member in complex with hemin has not been solved, site-directed mutational analysis identified Asn7, Trp67, and His77 as residues required for catalytic activity of S. aureus IsdG (55). This NWH triad is conserved in all identified IsdG family members (55).
Here we utilize genomic and biochemical techniques to identify and characterize a member of the IsdG family of heme-degrading monooxygenases from B. anthracis. In addition, the in vitro and in vivo hemin degradation activities of this protein are described. Finally, a role for IsdG in the pathogenesis of anthrax in an animal model of infection is investigated. This is the first description of the molecular machinery involved in heme uptake and degradation in B. anthracis.
MATERIALS AND METHODS
General methods and bacterial strains. Western blotting, transformations, plasmid purification, and subcloning were carried out as described previously (37). Deionized double-distilled water was used for all experiments. Oligonucleotides were synthesized by Integrated DNA Technologies. Escherichia coli strain DH5 [F– ara (lac-proAB) rpsL 80dlacZM15 hsdR17] was used for DNA manipulation, and E. coli strain BL21(DE3) [F– ompT hsdSB (rB– mB–) gal dcm (DE3)] was used for the expression of isdG. isdG was amplified by PCR using B. anthracis strain Sterne chromosomal DNA as a template. To prepare plasmid DNA for transformation into B. anthracis, vectors were passaged through the dam– E. coli strain K1077.
Bioinformatic analyses. BLAST analysis (1) was used to search the B. anthracis strain A2012 genome for genes homologous to the isd system of S. aureus (31). Individual protein comparisons were made using the MegAlign program from the LaserGene suite (DNASTAR, Madison, WI). Alignments and sequence distances were determined using the ClustalW method.
Construction of expression vectors. Expression vectors were constructed through PCR amplification of the complete isdG coding sequence. The subsequent PCR fragment was cloned into pCR2.1 (Invitrogen), and successful transformants were sequenced for accuracy. Inserts containing the correct sequence were subcloned into pET15B (Novagen) to create expression vectors containing an N-terminal histidine tag.
Mutagenesis of isdG. Sequences flanking isdG were amplified by PCR (primers sequences available upon request). Upstream and downstream fragments lacking an internal segment of isdG were cloned first into pCR2.1 (Invitrogen), with subsequent cloning into the chloramphenicol-resistant temperature-sensitive vector pTS1 (32). This vector was linearized at the junction of the upstream and downstream portions of isdG, and an ermC cassette was inserted. Inactivation of isdG was carried out by first passaging pTS1isdG::ermC through the restriction-deficient E. coli strain K1077. Plasmids were then extracted and electroporated into B. anthracis strain Sterne. B. anthracis strain Sterne isdG::ermC was created by passing B. anthracis strain Sterne containing pTS1 isdG::ermC in Luria-Bertani (LB) medium at the nonpermissive temperature in the presence of erythromycin for 3 days. Cultures were plated on prewarmed LB agar containing erythromycin and incubated overnight at 43°C. Colonies that arose were patched onto prewarmed LB agar containing either erythromycin or chloramphenicol and incubated at 42°C. Isolates that were resistant to erythromycin and sensitive to chloramphenicol were selected for further study. Successful inactivation of isdG was confirmed by PCR. Mutations were transduced into B. anthracis strain Sterne to eliminate the potential for secondary mutations. Bacteriophage CP-51 was used to infect the B. anthracis Sterne isdG::ermC strain. Plate lysates were generated and filtered. Filtered isdG::ermC CP-51 lysates were used to transduce the isdG::ermC allele into wild-type B. anthracis Sterne (described in reference 21). Erythromycin-resistant transductants were screened by PCR with isdG flanking and ermC-specific primers. PCR was used to verify the presence of the pXO-1 virulence plasmid.
Expression and purification of IsdG and IsdI. E. coli BL21(DE3) strains carrying pET15B-isdG were grown overnight at 37°C in LB medium containing 100 μg/ml ampicillin. The following day, cells were diluted into fresh medium and grown at 37°C to mid-log phase. At this time, the expression of the vectors was induced using 1 mM isopropyl-1-thiol-(D)-galactopyranoside. Cell growth was continued for 3 h at 30°C, and cells were harvested by centrifugation (10,000 x g for 15 min). Cells were lysed using a French press in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 100 μM phenylmethylsulfonyl fluoride. The cell suspension was centrifuged at 100,000 xg for 60 min. After centrifugation, the soluble fraction was applied to a Ni-nitrilotriacetic acid column, preequilibrated with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. The column was washed with 2 volumes of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl followed by a second washing with 3 volumes of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 10% glycerol and 10 mM imidazole. Protein was eluted in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 500 mM imidazole, and isolated fractions were dialyzed against 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. Purified protein was stored at –20°C.
Reconstitution of IsdG with hemin. The hemin-IsdG complex was prepared as described previously for hemin-heme oxygenase complexes (54, 58). Hemin and purified protein were mixed at a ratio of 3:1 (hemin/protein). The sample was applied to a nickel-nitrilotriacetic acid-agarose column preequilibrated with 50 mM Tris-HCl (pH 7.5), 100 mM NaCl. The column was then washed with the same buffer (3 volumes), and the protein was eluted in 500 mM imidazole. Fractions containing the heme-protein complexes were pooled and dialyzed against 50 mM Tris-HCl (pH 7.5), 100 mM NaCl.
Absorption spectroscopy. All absorption spectra were obtained using a Varian Cary 50BIO. Hemin binding studies were carried out by difference absorption spectroscopy in the Soret region. Aliquots of hemin (0.1 to 25 μM) were added to both the sample cuvette (10 μM IsdG) and reference cuvettes at 25°C. Spectra were recorded 5 min after the addition of hemin.
Hemin degradation assays. (i) Iron release assay. To measure the release of free iron from hemin, 10 μmol IsdG and 55 μl heme mixture (0.22 pmol [55Fe]hemin [RI Consultants] per 50 μl 2% bovine serum albumin) were placed into 50 mM HEPES, pH 7.4, 1 mM EDTA to a final volume of 1 ml and incubated at 30°C for 30 min. To quench the reaction, 10 μl 1 mM unlabeled hemin in 2% bovine serum albumin was added and samples were vortexed. Trichloroacetic acid was added to the sample at a concentration of 7.5% followed by incubation on ice for 30 min. The resulting precipitate was sedimented by centrifugation at 13,000 xg at 4°C for 15 min. A portion (750 μl) of the supernatant was withdrawn and added to 5 ml scintillation fluid. The amount of 55Fe was determined using a scintillation counter (Beckman LS600K).
(ii) Reaction with ascorbate. Ascorbic acid-dependent degradation of heme was monitored spectrophotometrically as previously described (59). IsdG-hemin (10 μM) in 50 mM Tris-HCl (pH 8.0) was incubated with ascorbic acid (10 mM), and the spectral changes between 300 and 800 nm were recorded every 5 min. The products of the reaction were extracted and subjected to high-pressure liquid chromatography (HPLC) as described below.
(iii) Reaction with NADPH-cytochrome P450 reductase. The reaction of IsdG-hemin in the presence of human NADPH-cytochrome P450 oxidoreductase (recombinant enzyme from Spodoptera frugiperda; Calbiochem) was similar to that previously described (54, 59). Human cytochrome P450 oxidoreductase was added to the IsdG-hemin complex (10 μM) at a ratio of reductase to Isd equal to 0.3:1 in a final volume of 1 ml 50 mM Tris-HCl (pH 8.0). Initiation of the reaction was carried out by the addition of NADPH in 10 μM increments to a final concentration of 100 μM. The spectral changes between 300 and 800 nm were monitored. Upon completion of the reaction, the reaction products were extracted and subjected to HPLC as described below.
(iv) Reaction in the presence of catalase. Purified recombinant catalase from Aspergillus niger (Sigma) was added to all reaction cuvettes at a ratio of catalase to hemoprotein equal to 0.5:1 immediately before the addition of either reductant or reductase.
HPLC separation of IsdG reaction products. Following the reaction of the hemin-IsdG complex with NADPH-cytochrome P450 oxidoreductase or ascorbate, 200 μl of glacial acetic acid and 200 μl of 3 M HCl were added to quench the cleavage reaction. The reaction mixture was then extracted into 1.5 ml chloroform. The organic layer was washed three times with 1 ml of distilled water, and the chloroform layer was removed under a stream of nitrogen. The resultant residue was dissolved in 800 μl of 85:15 (vol/vol) methanol-water prior to HPLC analysis. Samples were analyzed by reverse-phase HPLC on a Thermo hypersil C18 aquasil column (Keystone Scientific Operations), using a Beckman Coulter System Gold HPLC machine, eluted with 85:15 (vol/vol) methanol-water at a flow rate of 0.2 ml/min.
In vivo hemin utilization assay. To determine the role of IsdG in the utilization of hemin as an iron source in vivo, a plate assay was developed. B. anthracis cultures were grown in heart infusion broth (HIB) at 37°C overnight under the appropriate antibiotic selection. The following day, approximately 107 bacteria were plated on HIB agar, HIB agar containing 500 μM 2,2'-dipyridyl, or HIB agar containing 500 μM 2,2'-dipyridyl and various concentrations of hemin (0.5, 1.0, 0.5, 10, 20, and 50 μM). In the absence of added hemin, 500 μM 2,2'-dipyridyl completely inhibits the growth of all strains tested. After 48 h of incubation at 37°C in the dark, the number of CFU on HIB agar containing 2,2' dipyridyl and hemin was divided by the number of CFU on HIB agar and presented as the "hemin growth efficiency."
Hemin toxicity assay. To measure hemin toxicity, B. anthracis strains were grown to late log phase at 37°C in 5 ml of LB medium. Bacterial samples were removed, and approximately equal numbers of wild-type and isdG::ermC cells were inoculated into liquid LB medium containing various concentrations of hemin (0, 1, 5, 10, 20, 30, 40 μM). Bacteria were removed from hemin exposure at distinct time intervals (0, 1, 2, 3 h), washed, and serially diluted in LB medium. Dilutions were then plated on LB agar plates and incubated overnight at 37°C. The following day, the number of CFU present after exposure to various hemin concentrations was determined, compared to an unexposed cell population, and presented as the "survival rate."
Preparation of spores and animal infection model. Spores were prepared by growing B. anthracis strains in 2x SG medium at 37°C for 4 to 6 days until the majority of the culture had undergone sporulation, as determined by dark-field microscopy. After incubation, cultures were washed twice with double-distilled H2O and incubated for 60 min at 65°C to kill vegetative cells. Spores were then plated and checked for the pXO-1 virulence plasmid by colony PCR. For animal infections, 6- to 8-week-old A/J mice were infected subcutaneously with either 600 or 60,000 spores suspended in phosphate-buffered saline. Animals were monitored at 8-h intervals for clinical signs of disease. Moribund animals were euthanized with compressed CO2.
RESULTS
Identification of B. anthracis IsdG. The isd system of S. aureus encodes for a heme acquisition and import apparatus (30, 45). S. aureus Isd is composed of 10 genes (isdABCDEFGHI and srtB) encoding for cell wall-anchored hemoglobin (IsdB) (30) and hemoglobin-haptoglobin (IsdH/HarA) receptors (14), two cell wall-anchored heme binding proteins (IsdA, IsdC), a membrane transport system (IsdDEF) (30), two cytoplasmic heme-degrading monooxygenases of the IsdG family (IsdG, IsdI) (43, 55), and a transpeptidase (SrtB) (31). Using the nucleotide sequences of S. aureus isd genes as queries, a homologous system was identified in B. anthracis (Fig. 1A). This system is located in a single genomic region of the B. anthracis chromosome and encodes for proteins bearing identity to S. aureus IsdA, IsdC, IsdE, IsdF, IsdG, IsdI, and SrtB (Fig. 1A). Sequence analysis suggests that isdC, isdA1, isdA2, isdE, isdF, isdX, and srtB are transcribed in a uniform transcriptional direction, while isdG is independently expressed in a divergently transcribed cistron (Fig. 1A). Nucleotide sequences bearing similarity to Fur boxes, the consensus nucleotide sequence to which the iron-dependent repressor Fur binds (16), are located upstream of isdC and isdG, implying that these transcriptional units are upregulated under iron-starved conditions (Fig. 1C). The predicted protein product of B. anthracis IsdG is approximately 35% identical to S. aureus IsdG or IsdI across the entire length of the amino acid sequences. Furthermore, B. anthracis IsdG harbors the conserved N6, W67, and H76 (Fig. 1B), i.e., the catalytic NWH triad shown to be required for S. aureus IsdG-mediated hemin degradation (55). These results suggest that B. anthracis possesses an iron-regulated surface determinant (Isd) system encoding for a member of the heme-degrading IsdG family of monooxygenases.
Expression and purification of IsdG. IsdG was expressed as a six-histidine-tagged protein under the control of the T7 polymerase promoter in E. coli and purified by affinity chromatography on nickel-nitrilotriacetic acid-agarose, yielding a distinct protein band migrating at 12.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2A). This size corresponds well with the predicted size of monomeric IsdG. Typical purifications produced 15 mg of protein per liter of E. coli BL21(DE3).
Properties of the heme-IsdG complex. Absorption signals suggestive of hemin binding were not present upon spectral analysis of purified IsdG (Fig. 2B). Reconstitution of IsdG with hemin at pH 8.0 produced an optical absorption spectrum containing a Soret band at approximately 411 nm and a broad peak at approximately 550 nm (Fig. 2B), consistent with known heme binding proteins including those of S. aureus IsdG-hemin and IsdI-hemin (43). Spectrophotometric titration of IsdG with hemin can be used as a quantitative measure of interaction based on the fact that the Soret peak of the hemin-protein complexes is different than that of free hemin alone at neutral pH. Incremental addition of hemin to IsdG (10 μM) has a distinct inflection point at approximately 10 μM hemin, revealing a 1:1 stoichiometric relationship between protein and hemin (Fig. 2B, inset). These data were used to calculate molecular affinities using Michaelis-Menten kinetics, demonstrating that IsdG binds hemin with an equilibrium dissociation constant of 7.0 ± 1.2 μM. The pyridine hemochrome method was used to determine the millimolar extinction coefficient for IsdG to be 148 mM–1 cm–1. These values for extinction coefficient and dissociation constant are consistent with members of the IsdG-family of heme monooxygenases (43). Taken together, these results reveal that IsdG binds hemin in a manner resembling previously characterized members of the IsdG family of heme monooxygenases (55).
IsdG-mediated degradation of hemin to release free iron. To determine if IsdG is capable of releasing iron through cleavage of the porphyrin ring of hemin, we initially measured the ability of IsdG to enzymatically open the porphyrin macrocycle using optical absorption spectroscopy. In the presence of an electron donor in either the form of the reductant ascorbate or NADPH-cytochrome P450 reductase (53), heme oxygenases catalyze the oxidative degradation of hemin first to -mesohydroxyheme, followed by verdoheme, and finally, biliverdin (53). Each of these molecules exhibits a distinct color, allowing for spectrophotometric monitoring of enzymatic hemin degradation. Incubation of IsdG with hemin in the presence of either reductant or reductase leads to degradation of hemin (Fig. 3A and B). In the presence of ascorbic acid, this degradation proceeds to the formation of a product exhibiting an absorption spectrum consistent with that of the paradigmatic hemin degradation product biliverdin (Fig. 3B). Nonenzymatic degradation of heme can occur through a coupled oxidation reaction requiring exogenous hydrogen peroxide and can therefore be inhibited by catalase (2, 42). To distinguish between enzymatic degradation of hemin and the nonenzymatic coupled oxidation of hemin, the reactions were performed in the presence of exogenously added catalase. The presence of catalase did not significantly affect IsdG-mediated hemin degradation (Fig. 3A and B, inset), corroborating the conjecture that B. anthracis IsdG catalyzes the enzymatic degradation of hemin.
Enzymatic degradation of hemin to biliverdin is characterized by the opening of the macrocyclic conjunction of the porphyrin ring, and is therefore expected to lead to a concomitant release of free iron. To measure the IsdG-mediated release of free iron, a radiolabeled [55Fe]hemin molecule was incubated with IsdG in the presence of ascorbate and the amount of released 55Fe was recorded using a scintillation counter. This value was compared to iron release mediated by the S. aureus hemin binding protein IsdB, a protein that does not degrade hemin (30). Incubation of IsdG-hemin in the presence of reductant for 30 min resulted in an approximately fivefold increase in free iron release compared to that mediated by IsdB or buffer alone, confirming that free iron release occurs upon opening of the porphyrin macrocycle (Fig. 3C). To verify that the degradation product of ascorbate-catalyzed IsdG-mediated hemin degradation was biliverdin, elution profiles of products from the B. anthracis IsdG (BaIsdG)-catalyzed reaction were compared to the elution profiles of the products from S. aureus IsdG (SaIsdG)-catalyzed hemin degradation using HPLC. The products from both of these reactions had similar elution profiles (Fig. 4) implying that B. anthracis IsdG degrades hemin to produce biliverdin, as has been reported for the S. aureus IsdG and IsdI enzymes (43). These results demonstrate that B. anthracis IsdG enzymatically degrades hemin to produce free iron and biliverdin.
IsdG is required for hemin utilization and protection against hemin-mediated toxicity in B. anthracis. To determine whether IsdG is required for the utilization of hemin as an iron source, isdG was inactivated through allelic replacement, and mutant bacteria were subjected to a plate-based hemin utilization assay. In this assay, wild-type B. anthracis strain Sterne is inhibited for growth at hemin concentrations below 1 μM and above 5 μM (Fig. 5), presumably due to iron starvation and heme toxicity, respectively. Inactivation of isdG eliminated growth of B. anthracis below 1 μM hemin and significantly increased the hemin-mediated toxicity experienced by this strain at concentrations above 5 μM hemin (Fig. 5). Inactivation of isdG did not affect the ability of B. anthracis to grow on medium containing nonhemin iron sources (data not shown). The measurable growth of isdG::ermC between 1 to 5 μM hemin may be due to the presence of contaminating free iron in the hemin preparations utilized. Low amounts of contaminating free iron in the hemin preparation would relieve iron starvation of a heme oxygenase mutant; however, this contaminating iron would not be expected to affect hemin-mediated toxicity. Alternatively, the growth of the isdG mutant strain between 1 to 5 μM hemin could be due to the presence of additional as-yet-unidentified heme-degrading enzymes in B. anthracis.
To expand on the observation that inactivation of isdG increases the sensitivity of B. anthracis to hemin, a hemin toxicity assay was developed. In this assay, exposure of B. anthracis to concentrations of hemin above 1 μM led to hemin-mediated toxicity after 1 h (Fig. 6). This toxicity is concentration dependent and becomes more pronounced over time, with concentrations of hemin above 30 μM eliminating detectable B. anthracis strain Sterne after 2 h. Inactivation of isdG significantly increases hemin-mediated toxicity to the point where, after 1 h, we were unable to detect live bacteria upon exposure of B. anthracis isdG::ermC to concentrations of hemin above 10 μM (Fig. 6). These results demonstrate that IsdG not only degrades hemin to release free iron as a nutrient source but also protects the bacterium against the damaging effects caused by the accumulation of intracellular hemin.
The observation that isdG is required to protect against hemin-mediated toxicity suggests that IsdG is involved in protecting B. anthracis against reactive oxygen species generated upon exposure to high amounts of hemin. If this were the case, the hemin-mediated toxicity might be augmented by the respiratory burst of activated macrophages. To test this hypothesis, wild-type and isdG::ermC strains were measured for their ability to survive and grow in activated macrophages. We did not identify differences in the growth rate of wild-type and isdG::ermC strains in activated macrophages (data not shown). These results demonstrate that hemin degradation does not play a role in growth inside activated macrophages, suggesting that hemin degradation is important at sites of infection outside the macrophage-associated replication cycle of B. anthracis.
Role of IsdG in virulence during A/J mouse infections. B. anthracis secretes lethal toxin and edema toxin to cause anthrax disease (11). Three pXO1 virulence plasmid genes encode subunits for both toxins, pag (protective antigen [PA]), lef (lethal factor), and cya (edema factor), as PA performs binding and host cell transport functions for both lethal factor and edema factor (11). The toxin genes are known to be essential for disease progression in multiple animal models of anthrax cutaneous infection, including guinea pigs and A/J mice (5, 6, 34). Further, antibodies against PA appear to be a critical factor in protective immunity (26, 50). B. anthracis strain Sterne lacks the pXO2 virulence plasmid, encoding the capABCD genes responsible for synthesis of the poly-D-glutamic acid capsule (17, 33, 46). The glutamate capsule of B. anthracis is essential for the pathogenesis of cutaneous anthrax infections in mice and, presumably, in many other animal infections (15); however, strain Sterne retains significant virulence in the A/J mouse model, as these animals display significant defects in phagocytic killing of bacterial pathogens (51, 52).
B. anthracis strain Ames 50% lethal doses of 50 spores are required for the development of lethal anthrax disease in mice (34), whereas a subcutaneous 50% lethal dose of 106 Sterne spores is required to generate a similar disease (34). Welkos and colleagues showed that subcutaneous infection of A/J mice with B. anthracis strain Sterne spores leads to an acute lethal disease at a dose of 102 to 103 spores (51). Subcutaneous infection of A/J mice with 60,000 spores of either B. anthracis strain Sterne or isdG::ermC killed 100% of the animals within 3 days of infection. In addition, low-dose infection with 600 spores of either B. anthracis strain Sterne or isdG::ermC caused a lethal infection in 40 to 50% of the animals by 5 days postinfection (Fig. 7). These results suggest that IsdG-mediated heme degradation is not required for the ability of B. anthracis strain Sterne to cause systemic anthrax in the A/J mouse model of infection.
DISCUSSION
As observed for most bacterial pathogens, B. anthracis requires iron to multiply and to initiate and maintain pathogenic processes that lead to anthrax disease. The complex nature of the B. anthracis life cycle, combined with multiple potential entry points into the host, presumably necessitates diverse iron acquisition systems in this organism. Early in infection, the anthrax spore is presumed to be engulfed by circulating macrophages. After engulfment, B. anthracis spores germinate inside the macrophage, requiring iron for the vegetative cells to multiple and divide (13, 22, 23). Heme-containing proteins inside the macrophage are not predicted to be in abundance, and presumably reside primarily in host membranes, making them unlikely iron sources for intracellular bacilli. Due to the unavailability of intracellular heme, it seems more likely that the labile iron pool (8) or iron complexed to iron mobilization or storage proteins such as transferrin or ferritin is the primary iron source utilized during the intracellular life cycle of B. anthracis. These host iron sources are believed to be the primary target of bacterial siderophores, potentially explaining the severe decrease in virulence exhibited by a B. anthracis siderophore synthesis mutant (9). Once B. anthracis lyses the macrophage and enters the host bloodstream, additional iron sources are likely required for infection to progress. The most abundant iron source in the blood of mammalian hosts is in the form of hemoproteins such as hemoglobin (12). To liberate and utilize hemoglobin as an iron source, bacterial pathogens must have a mechanism capable of lysing the host's red blood cells. In the case of B. anthracis, this lysis might be mediated by cytolysins such as the cholesterol-dependent anthrolysin O (41). Once hemoglobin is liberated from lysed erythrocytes, systems responsible for hemoglobin binding, heme removal and transport into the cytoplasm, and heme degradation may be required for the utilization of heme as an iron source.
The isd system has been shown to be responsible for a portion of the binding and transport of heme iron into the cytoplasm of S. aureus (30). Homologous systems have been identified in numerous gram-positive pathogens, including Clostridium tetani, Listeria monocytogenes, and B. anthracis, implying that a conserved mechanism of heme binding, transport, and utilization exists in this group of human pathogens (45). As has been shown in S. aureus, B. anthracis presumably utilizes the Isd system to recognize host hemoproteins, transport heme into the bacterial cytoplasm, and degrade heme to release free iron. Two proteins of the staphylococcal Isd system, IsdG and IsdI, are monooxygenases responsible for the degradation of heme (43). IsdG and IsdI are members of the IsdG family of heme monooxygenases characterized by a -barrel type fold, assembled from the IsdG homodimer with two catalytic sites defined by the conserved NWH triad (55). Initial biochemical and structural characterization of the staphylococcal members of the IsdG family have been performed; however, the role of IsdG family-mediated heme degradation in the biology and virulence of a gram-positive pathogen has not been reported.
Here we show that B. anthracis expresses an IsdG family member possessing the conserved NWH triad that is also involved in hemin degradation. B. anthracis IsdG degrades hemin in vitro and in vivo to release free iron. The degradation product of BaIsdG-mediated hemin degradation has an HPLC elution profile similar to that of the degradation products produced upon SaIsdG- and SaIsdI-mediated hemin degradation, implying that all of these enzymes catalyze the degradation of hemin to release free iron and form bilverdin (43). Inactivation of B. anthracis isdG leads to an inability to utilize low amounts of hemin as a sole iron source and also prevents the ability of B. anthracis to grow in the presence of high levels of hemin (Fig. 5 and 6). This heme-mediated toxicity suggests that not only is IsdG-mediated heme degradation necessary for the utilization of hemin as an iron source, it is also involved in protection against the toxic effects of heme caused via the buildup of oxygen radicals. Ascribing a protective function to a heme oxygenase is not without precedence, as a primary function of vertebrate heme oxygenases is protection of the host against the toxic effects of heme (10, 24) and an effect of heme oxygenases on heme-mediated toxicity has been reported in the genus Neisseria (59). Inactivation of B. anthracis isdG did not affect the ability of the organism to proliferate inside the macrophage cell line J774, suggesting that heme degradation may not play a role in macrophage replication of B. anthracis. However, inactivation of srtB decreases growth of B. anthracis in the mouse macrophage-like cell line J774A.1 (60), suggesting that a functional heme uptake system is required for survival in this host niche. It is possible that the loss of IsdG is compensated for by additional enzymes in B. anthracis that are capable of degrading heme. Further studies are needed to delineate between the requirement for IsdG-mediated heme degradation in acquisition of iron as a nutrient and protection against heme-mediated toxicity.
Inactivation of isdG did not decrease the ability of B. anthracis strain Sterne to mediate disease in the A/J mouse model of systemic infection. A number of possible mechanisms explain this lack of an effect on virulence. Based on a strict requirement for siderophore-acquired iron in B. anthracis pathogenesis (9), it is possible that the infecting bacillus satisfies its iron requirement through siderophore-mediated acquisition of nonheme iron. This would imply that heme iron acquisition is not a prominent iron acquisition strategy during the pathogenesis of anthrax. Alternatively, the Sterne-A/J infection model might not adequately represent the infectious process inside a human. This possibility is supported by the absence of the pXO-2 virulence plasmid in B. anthracis strain Sterne and the immune deficiency of the A/J mouse strain (15, 51). We favor the hypothesis that additional heme-degrading enzymes exist in B. anthracis that have yet to be identified. This supposition is based largely on the measurable growth of the isdG::ermC strain at hemin concentrations between 1 and 5 μM. Multiple heme-degrading enzymes within a single organism have been reported for Pseudomonas aeruginosa (49) and S. aureus (43), and a recent genomic analysis of bacterial heme oxygenases proposed the existence of distinct heme-degrading enzyme families in bacteria (18). Using genomic analyses, we have been unable to identify any additional candidate heme oxygenases in the genome of B. anthracis, including those of the HO family (hmuO [38], pigA [35], hemO [59]) or the IsdG family (isdG, isdI [43]). In addition, we have not identified any potential members of the hutZ/shuS heme iron utilization genes (56, 57) in the B. anthracis genome. Together, these observations support the possibility that members of an additional, as yet unidentified heme-degrading enzyme family exist in B. anthracis.
Based on sequence analysis and the results shown here, we hypothesize that heme iron is acquired through the B. anthracis Isd system. In our model, hemoproteins are recognized by the putative cell wall-anchored proteins IsdA and IsdC and heme is removed and transported through the IsdEFX membrane transport system. Once inside the cytoplasm, heme is degraded by IsdG acting as a heme monooxygenase to release free iron and form biliverdin. IsdG-mediated heme degradation can provide nutrient iron to the rapidly multiplying bacillus as well as protect B. anthracis from heme-mediated toxicity that might be encountered upon exposure to oxidative radicals. A lack of a role for IsdG in growth inside an activated macrophage, combined with a requirement for IsdG to protect against heme-mediated toxicity, suggests that this activity might be important for protecting the bacterium against the large amounts of endogenous reactive oxygen species that would be formed during the cellular metabolism associated with the rapid microbial expansion that is characteristic of anthrax (29).
The demonstrated importance of heme iron during infection of other gram-positive pathogens (44), combined with the lack of a role for IsdG in the pathogenesis of B. anthracis reported here, supports the notion that additional heme-degrading enzymes exist in this organism. Recent advances in the availability of B. anthracis genome sequences (36), combined with an increased appreciation for the role of bacterial heme oxygenases in iron acquisition (18), will contribute greatly to our ability to further investigate the possibility that B. anthracis encodes multiple heme-degrading enzymes.
ACKNOWLEDGMENTS
We thank members of the Schneewind laboratory for critical reading of the manuscript as well as Joshua M. Barnett, Christina Tam, Luciano Marraffini, and Kristin DeBord for experimental assistance in the course of these studies.
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ABSTRACT
Bacillus anthracis, the causative agent of anthrax, utilizes hemin and hemoglobin for growth in culture, suggesting that these host molecules serve as sources for the nutrient iron during bacterial infection. Bioinformatic analyses of the B. anthracis genome revealed genes with similarity to the iron-regulated surface determinant (isd) system responsible for heme uptake in Staphylococcus aureus. We show that the protein product of one of these genes, isdG, binds hemin in a manner resembling the heme binding of known heme oxygenases. Formation of IsdG:hemin complexes in the presence of a suitable electron donor, e.g., ascorbate or cytochrome P450 reductase, promotes catalytic degradation of hemin to biliverdin with concomitant release of iron. IsdG is required for B. anthracis utilization of hemin as a sole iron source, and it is also necessary for bacterial protection against heme-mediated toxicity. These data suggest that IsdG functions as a heme-degrading monooxygenase in B. anthracis.
INTRODUCTION
The ability to acquire iron during infection is an essential attribute for many bacterial pathogens of vertebrates (4). This is due to the requirement for iron in numerous cellular processes that are fundamental for life, such as DNA replication, energy generation, and protection against reactive oxygen species (4). A primary human defense mechanism against bacterial infection is to severely limit available iron through the production of iron binding proteins. The most abundant group of iron binding proteins in humans are hemoproteins, polypeptides that utilize heme as a cofactor (12). Heme, a tetrapyrrole encircling an individual iron atom within the macrocyclic conjunction of the heme porphyrin ring, and hemoproteins represent approximately 80% of the iron in humans or animals (12). To successfully mediate infection, most bacterial pathogens have developed systems capable of extracting heme from human hemoproteins and subsequently utilizing this cofactor as both a heme and iron source (4, 7, 20, 45, 48). The molecular mechanisms whereby gram-negative bacterial pathogens acquire iron from heme are well characterized (4, 7, 20, 48). In contrast, the molecular mechanisms involved in heme iron scavenging in gram-positive pathogens are only now beginning to emerge (3, 28, 30, 45, 54). Nevertheless, heme has been shown to be the preferred source of iron during the pathogenesis of the gram-positive pathogen Staphylococcus aureus (44).
Bacillus anthracis is a gram-positive spore-forming rod and the causative agent of the disease anthrax (27). The systemic form of anthrax begins upon engulfment of the bacterial spores by resident macrophages, followed by intracellular germination and subsequent replication and amplification of the vegetative form of bacilli (13, 22, 23). Ultimately, vegetative cells of B. anthracis escape macrophages and enter circulation where they multiply in the bloodstream of infected individuals, at times reaching cell densities as high as 109 per ml (29). This large amount of growth necessitates a rapid accumulation of nutrients for the organism to satisfy its nutritional needs during replication. The only nutrient thought to be limiting for growth of bacterial pathogens during infection is iron (8), suggesting that efficient mechanisms for iron acquisition exist in B. anthracis. This hypothesis is corroborated by the presence of 15 putative ATP-binding cassette-type uptake systems specific for iron and two gene clusters exhibiting homologies consistent with siderophore synthesis encoded in the genome of B. anthracis (36). The production of two separate siderophores has been experimentally confirmed in this bacterium (9, 19), one of which is required for virulence in a mouse model of systemic infection (9). In addition to these siderophores, B. anthracis is capable of using heme, hemoproteins, iron-transferrin, and certain heterologous siderophores (xenosiderophores) as iron sources for growth (19). The molecular mechanisms responsible for the utilization of these iron sources are as yet unknown.
For bacterial pathogens to utilize heme as a nutrient source, the heme-bound iron atom must be liberated through opening of the porphyrin ring. Structural comparisons among known heme oxygenases identified two enzyme families capable of degrading heme to biliverdin and liberating iron in the presence of a suitable electron donor. The first is catalyzed by members of the heme oxygenase family, a ubiquitous enzyme family whose members have been identified in all kingdoms, including the bacterial pathogens Neisseria gonorrhoeae (59), Neisseria meningitidis (59), Pseudomonas aeruginosa (35), Corynebacterium diphtheria, and Corynebacterium ulcerans (38). Heme oxygenase family members are characterized by a conserved histidine residue in the N-terminal portion of the protein and a GXXXG motif required for catalytic activity (53).
Crystal structure analyses revealed that heme oxygenase family members function as monomeric alpha helical proteins with a single active site (25, 39, 40, 47, 53). The second family of heme-degrading monooxygenases, the IsdG family, is found exclusively in gram-positive bacteria (43, 45, 55). The genome of S. aureus encodes two IsdG family members, IsdG and IsdI, which are part of the iron-regulated surface determinant (isd) system of heme uptake (30). These enzymes are responsible for the degradation of hemin to free iron and biliverdin in the presence of an electron donor in vitro and confer the ability to utilize hemin as a sole iron source on a heme oxygenase mutant of C. ulcerans (43). The crystal structures of S. aureus IsdG and IsdI demonstrate that these enzymes fold in a dimeric beta-barrel type configuration encompassing two separate active sites for heme degradation (55). Although the crystal structure of an IsdG family member in complex with hemin has not been solved, site-directed mutational analysis identified Asn7, Trp67, and His77 as residues required for catalytic activity of S. aureus IsdG (55). This NWH triad is conserved in all identified IsdG family members (55).
Here we utilize genomic and biochemical techniques to identify and characterize a member of the IsdG family of heme-degrading monooxygenases from B. anthracis. In addition, the in vitro and in vivo hemin degradation activities of this protein are described. Finally, a role for IsdG in the pathogenesis of anthrax in an animal model of infection is investigated. This is the first description of the molecular machinery involved in heme uptake and degradation in B. anthracis.
MATERIALS AND METHODS
General methods and bacterial strains. Western blotting, transformations, plasmid purification, and subcloning were carried out as described previously (37). Deionized double-distilled water was used for all experiments. Oligonucleotides were synthesized by Integrated DNA Technologies. Escherichia coli strain DH5 [F– ara (lac-proAB) rpsL 80dlacZM15 hsdR17] was used for DNA manipulation, and E. coli strain BL21(DE3) [F– ompT hsdSB (rB– mB–) gal dcm (DE3)] was used for the expression of isdG. isdG was amplified by PCR using B. anthracis strain Sterne chromosomal DNA as a template. To prepare plasmid DNA for transformation into B. anthracis, vectors were passaged through the dam– E. coli strain K1077.
Bioinformatic analyses. BLAST analysis (1) was used to search the B. anthracis strain A2012 genome for genes homologous to the isd system of S. aureus (31). Individual protein comparisons were made using the MegAlign program from the LaserGene suite (DNASTAR, Madison, WI). Alignments and sequence distances were determined using the ClustalW method.
Construction of expression vectors. Expression vectors were constructed through PCR amplification of the complete isdG coding sequence. The subsequent PCR fragment was cloned into pCR2.1 (Invitrogen), and successful transformants were sequenced for accuracy. Inserts containing the correct sequence were subcloned into pET15B (Novagen) to create expression vectors containing an N-terminal histidine tag.
Mutagenesis of isdG. Sequences flanking isdG were amplified by PCR (primers sequences available upon request). Upstream and downstream fragments lacking an internal segment of isdG were cloned first into pCR2.1 (Invitrogen), with subsequent cloning into the chloramphenicol-resistant temperature-sensitive vector pTS1 (32). This vector was linearized at the junction of the upstream and downstream portions of isdG, and an ermC cassette was inserted. Inactivation of isdG was carried out by first passaging pTS1isdG::ermC through the restriction-deficient E. coli strain K1077. Plasmids were then extracted and electroporated into B. anthracis strain Sterne. B. anthracis strain Sterne isdG::ermC was created by passing B. anthracis strain Sterne containing pTS1 isdG::ermC in Luria-Bertani (LB) medium at the nonpermissive temperature in the presence of erythromycin for 3 days. Cultures were plated on prewarmed LB agar containing erythromycin and incubated overnight at 43°C. Colonies that arose were patched onto prewarmed LB agar containing either erythromycin or chloramphenicol and incubated at 42°C. Isolates that were resistant to erythromycin and sensitive to chloramphenicol were selected for further study. Successful inactivation of isdG was confirmed by PCR. Mutations were transduced into B. anthracis strain Sterne to eliminate the potential for secondary mutations. Bacteriophage CP-51 was used to infect the B. anthracis Sterne isdG::ermC strain. Plate lysates were generated and filtered. Filtered isdG::ermC CP-51 lysates were used to transduce the isdG::ermC allele into wild-type B. anthracis Sterne (described in reference 21). Erythromycin-resistant transductants were screened by PCR with isdG flanking and ermC-specific primers. PCR was used to verify the presence of the pXO-1 virulence plasmid.
Expression and purification of IsdG and IsdI. E. coli BL21(DE3) strains carrying pET15B-isdG were grown overnight at 37°C in LB medium containing 100 μg/ml ampicillin. The following day, cells were diluted into fresh medium and grown at 37°C to mid-log phase. At this time, the expression of the vectors was induced using 1 mM isopropyl-1-thiol-(D)-galactopyranoside. Cell growth was continued for 3 h at 30°C, and cells were harvested by centrifugation (10,000 x g for 15 min). Cells were lysed using a French press in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 100 μM phenylmethylsulfonyl fluoride. The cell suspension was centrifuged at 100,000 xg for 60 min. After centrifugation, the soluble fraction was applied to a Ni-nitrilotriacetic acid column, preequilibrated with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. The column was washed with 2 volumes of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl followed by a second washing with 3 volumes of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 10% glycerol and 10 mM imidazole. Protein was eluted in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl containing 500 mM imidazole, and isolated fractions were dialyzed against 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. Purified protein was stored at –20°C.
Reconstitution of IsdG with hemin. The hemin-IsdG complex was prepared as described previously for hemin-heme oxygenase complexes (54, 58). Hemin and purified protein were mixed at a ratio of 3:1 (hemin/protein). The sample was applied to a nickel-nitrilotriacetic acid-agarose column preequilibrated with 50 mM Tris-HCl (pH 7.5), 100 mM NaCl. The column was then washed with the same buffer (3 volumes), and the protein was eluted in 500 mM imidazole. Fractions containing the heme-protein complexes were pooled and dialyzed against 50 mM Tris-HCl (pH 7.5), 100 mM NaCl.
Absorption spectroscopy. All absorption spectra were obtained using a Varian Cary 50BIO. Hemin binding studies were carried out by difference absorption spectroscopy in the Soret region. Aliquots of hemin (0.1 to 25 μM) were added to both the sample cuvette (10 μM IsdG) and reference cuvettes at 25°C. Spectra were recorded 5 min after the addition of hemin.
Hemin degradation assays. (i) Iron release assay. To measure the release of free iron from hemin, 10 μmol IsdG and 55 μl heme mixture (0.22 pmol [55Fe]hemin [RI Consultants] per 50 μl 2% bovine serum albumin) were placed into 50 mM HEPES, pH 7.4, 1 mM EDTA to a final volume of 1 ml and incubated at 30°C for 30 min. To quench the reaction, 10 μl 1 mM unlabeled hemin in 2% bovine serum albumin was added and samples were vortexed. Trichloroacetic acid was added to the sample at a concentration of 7.5% followed by incubation on ice for 30 min. The resulting precipitate was sedimented by centrifugation at 13,000 xg at 4°C for 15 min. A portion (750 μl) of the supernatant was withdrawn and added to 5 ml scintillation fluid. The amount of 55Fe was determined using a scintillation counter (Beckman LS600K).
(ii) Reaction with ascorbate. Ascorbic acid-dependent degradation of heme was monitored spectrophotometrically as previously described (59). IsdG-hemin (10 μM) in 50 mM Tris-HCl (pH 8.0) was incubated with ascorbic acid (10 mM), and the spectral changes between 300 and 800 nm were recorded every 5 min. The products of the reaction were extracted and subjected to high-pressure liquid chromatography (HPLC) as described below.
(iii) Reaction with NADPH-cytochrome P450 reductase. The reaction of IsdG-hemin in the presence of human NADPH-cytochrome P450 oxidoreductase (recombinant enzyme from Spodoptera frugiperda; Calbiochem) was similar to that previously described (54, 59). Human cytochrome P450 oxidoreductase was added to the IsdG-hemin complex (10 μM) at a ratio of reductase to Isd equal to 0.3:1 in a final volume of 1 ml 50 mM Tris-HCl (pH 8.0). Initiation of the reaction was carried out by the addition of NADPH in 10 μM increments to a final concentration of 100 μM. The spectral changes between 300 and 800 nm were monitored. Upon completion of the reaction, the reaction products were extracted and subjected to HPLC as described below.
(iv) Reaction in the presence of catalase. Purified recombinant catalase from Aspergillus niger (Sigma) was added to all reaction cuvettes at a ratio of catalase to hemoprotein equal to 0.5:1 immediately before the addition of either reductant or reductase.
HPLC separation of IsdG reaction products. Following the reaction of the hemin-IsdG complex with NADPH-cytochrome P450 oxidoreductase or ascorbate, 200 μl of glacial acetic acid and 200 μl of 3 M HCl were added to quench the cleavage reaction. The reaction mixture was then extracted into 1.5 ml chloroform. The organic layer was washed three times with 1 ml of distilled water, and the chloroform layer was removed under a stream of nitrogen. The resultant residue was dissolved in 800 μl of 85:15 (vol/vol) methanol-water prior to HPLC analysis. Samples were analyzed by reverse-phase HPLC on a Thermo hypersil C18 aquasil column (Keystone Scientific Operations), using a Beckman Coulter System Gold HPLC machine, eluted with 85:15 (vol/vol) methanol-water at a flow rate of 0.2 ml/min.
In vivo hemin utilization assay. To determine the role of IsdG in the utilization of hemin as an iron source in vivo, a plate assay was developed. B. anthracis cultures were grown in heart infusion broth (HIB) at 37°C overnight under the appropriate antibiotic selection. The following day, approximately 107 bacteria were plated on HIB agar, HIB agar containing 500 μM 2,2'-dipyridyl, or HIB agar containing 500 μM 2,2'-dipyridyl and various concentrations of hemin (0.5, 1.0, 0.5, 10, 20, and 50 μM). In the absence of added hemin, 500 μM 2,2'-dipyridyl completely inhibits the growth of all strains tested. After 48 h of incubation at 37°C in the dark, the number of CFU on HIB agar containing 2,2' dipyridyl and hemin was divided by the number of CFU on HIB agar and presented as the "hemin growth efficiency."
Hemin toxicity assay. To measure hemin toxicity, B. anthracis strains were grown to late log phase at 37°C in 5 ml of LB medium. Bacterial samples were removed, and approximately equal numbers of wild-type and isdG::ermC cells were inoculated into liquid LB medium containing various concentrations of hemin (0, 1, 5, 10, 20, 30, 40 μM). Bacteria were removed from hemin exposure at distinct time intervals (0, 1, 2, 3 h), washed, and serially diluted in LB medium. Dilutions were then plated on LB agar plates and incubated overnight at 37°C. The following day, the number of CFU present after exposure to various hemin concentrations was determined, compared to an unexposed cell population, and presented as the "survival rate."
Preparation of spores and animal infection model. Spores were prepared by growing B. anthracis strains in 2x SG medium at 37°C for 4 to 6 days until the majority of the culture had undergone sporulation, as determined by dark-field microscopy. After incubation, cultures were washed twice with double-distilled H2O and incubated for 60 min at 65°C to kill vegetative cells. Spores were then plated and checked for the pXO-1 virulence plasmid by colony PCR. For animal infections, 6- to 8-week-old A/J mice were infected subcutaneously with either 600 or 60,000 spores suspended in phosphate-buffered saline. Animals were monitored at 8-h intervals for clinical signs of disease. Moribund animals were euthanized with compressed CO2.
RESULTS
Identification of B. anthracis IsdG. The isd system of S. aureus encodes for a heme acquisition and import apparatus (30, 45). S. aureus Isd is composed of 10 genes (isdABCDEFGHI and srtB) encoding for cell wall-anchored hemoglobin (IsdB) (30) and hemoglobin-haptoglobin (IsdH/HarA) receptors (14), two cell wall-anchored heme binding proteins (IsdA, IsdC), a membrane transport system (IsdDEF) (30), two cytoplasmic heme-degrading monooxygenases of the IsdG family (IsdG, IsdI) (43, 55), and a transpeptidase (SrtB) (31). Using the nucleotide sequences of S. aureus isd genes as queries, a homologous system was identified in B. anthracis (Fig. 1A). This system is located in a single genomic region of the B. anthracis chromosome and encodes for proteins bearing identity to S. aureus IsdA, IsdC, IsdE, IsdF, IsdG, IsdI, and SrtB (Fig. 1A). Sequence analysis suggests that isdC, isdA1, isdA2, isdE, isdF, isdX, and srtB are transcribed in a uniform transcriptional direction, while isdG is independently expressed in a divergently transcribed cistron (Fig. 1A). Nucleotide sequences bearing similarity to Fur boxes, the consensus nucleotide sequence to which the iron-dependent repressor Fur binds (16), are located upstream of isdC and isdG, implying that these transcriptional units are upregulated under iron-starved conditions (Fig. 1C). The predicted protein product of B. anthracis IsdG is approximately 35% identical to S. aureus IsdG or IsdI across the entire length of the amino acid sequences. Furthermore, B. anthracis IsdG harbors the conserved N6, W67, and H76 (Fig. 1B), i.e., the catalytic NWH triad shown to be required for S. aureus IsdG-mediated hemin degradation (55). These results suggest that B. anthracis possesses an iron-regulated surface determinant (Isd) system encoding for a member of the heme-degrading IsdG family of monooxygenases.
Expression and purification of IsdG. IsdG was expressed as a six-histidine-tagged protein under the control of the T7 polymerase promoter in E. coli and purified by affinity chromatography on nickel-nitrilotriacetic acid-agarose, yielding a distinct protein band migrating at 12.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 2A). This size corresponds well with the predicted size of monomeric IsdG. Typical purifications produced 15 mg of protein per liter of E. coli BL21(DE3).
Properties of the heme-IsdG complex. Absorption signals suggestive of hemin binding were not present upon spectral analysis of purified IsdG (Fig. 2B). Reconstitution of IsdG with hemin at pH 8.0 produced an optical absorption spectrum containing a Soret band at approximately 411 nm and a broad peak at approximately 550 nm (Fig. 2B), consistent with known heme binding proteins including those of S. aureus IsdG-hemin and IsdI-hemin (43). Spectrophotometric titration of IsdG with hemin can be used as a quantitative measure of interaction based on the fact that the Soret peak of the hemin-protein complexes is different than that of free hemin alone at neutral pH. Incremental addition of hemin to IsdG (10 μM) has a distinct inflection point at approximately 10 μM hemin, revealing a 1:1 stoichiometric relationship between protein and hemin (Fig. 2B, inset). These data were used to calculate molecular affinities using Michaelis-Menten kinetics, demonstrating that IsdG binds hemin with an equilibrium dissociation constant of 7.0 ± 1.2 μM. The pyridine hemochrome method was used to determine the millimolar extinction coefficient for IsdG to be 148 mM–1 cm–1. These values for extinction coefficient and dissociation constant are consistent with members of the IsdG-family of heme monooxygenases (43). Taken together, these results reveal that IsdG binds hemin in a manner resembling previously characterized members of the IsdG family of heme monooxygenases (55).
IsdG-mediated degradation of hemin to release free iron. To determine if IsdG is capable of releasing iron through cleavage of the porphyrin ring of hemin, we initially measured the ability of IsdG to enzymatically open the porphyrin macrocycle using optical absorption spectroscopy. In the presence of an electron donor in either the form of the reductant ascorbate or NADPH-cytochrome P450 reductase (53), heme oxygenases catalyze the oxidative degradation of hemin first to -mesohydroxyheme, followed by verdoheme, and finally, biliverdin (53). Each of these molecules exhibits a distinct color, allowing for spectrophotometric monitoring of enzymatic hemin degradation. Incubation of IsdG with hemin in the presence of either reductant or reductase leads to degradation of hemin (Fig. 3A and B). In the presence of ascorbic acid, this degradation proceeds to the formation of a product exhibiting an absorption spectrum consistent with that of the paradigmatic hemin degradation product biliverdin (Fig. 3B). Nonenzymatic degradation of heme can occur through a coupled oxidation reaction requiring exogenous hydrogen peroxide and can therefore be inhibited by catalase (2, 42). To distinguish between enzymatic degradation of hemin and the nonenzymatic coupled oxidation of hemin, the reactions were performed in the presence of exogenously added catalase. The presence of catalase did not significantly affect IsdG-mediated hemin degradation (Fig. 3A and B, inset), corroborating the conjecture that B. anthracis IsdG catalyzes the enzymatic degradation of hemin.
Enzymatic degradation of hemin to biliverdin is characterized by the opening of the macrocyclic conjunction of the porphyrin ring, and is therefore expected to lead to a concomitant release of free iron. To measure the IsdG-mediated release of free iron, a radiolabeled [55Fe]hemin molecule was incubated with IsdG in the presence of ascorbate and the amount of released 55Fe was recorded using a scintillation counter. This value was compared to iron release mediated by the S. aureus hemin binding protein IsdB, a protein that does not degrade hemin (30). Incubation of IsdG-hemin in the presence of reductant for 30 min resulted in an approximately fivefold increase in free iron release compared to that mediated by IsdB or buffer alone, confirming that free iron release occurs upon opening of the porphyrin macrocycle (Fig. 3C). To verify that the degradation product of ascorbate-catalyzed IsdG-mediated hemin degradation was biliverdin, elution profiles of products from the B. anthracis IsdG (BaIsdG)-catalyzed reaction were compared to the elution profiles of the products from S. aureus IsdG (SaIsdG)-catalyzed hemin degradation using HPLC. The products from both of these reactions had similar elution profiles (Fig. 4) implying that B. anthracis IsdG degrades hemin to produce biliverdin, as has been reported for the S. aureus IsdG and IsdI enzymes (43). These results demonstrate that B. anthracis IsdG enzymatically degrades hemin to produce free iron and biliverdin.
IsdG is required for hemin utilization and protection against hemin-mediated toxicity in B. anthracis. To determine whether IsdG is required for the utilization of hemin as an iron source, isdG was inactivated through allelic replacement, and mutant bacteria were subjected to a plate-based hemin utilization assay. In this assay, wild-type B. anthracis strain Sterne is inhibited for growth at hemin concentrations below 1 μM and above 5 μM (Fig. 5), presumably due to iron starvation and heme toxicity, respectively. Inactivation of isdG eliminated growth of B. anthracis below 1 μM hemin and significantly increased the hemin-mediated toxicity experienced by this strain at concentrations above 5 μM hemin (Fig. 5). Inactivation of isdG did not affect the ability of B. anthracis to grow on medium containing nonhemin iron sources (data not shown). The measurable growth of isdG::ermC between 1 to 5 μM hemin may be due to the presence of contaminating free iron in the hemin preparations utilized. Low amounts of contaminating free iron in the hemin preparation would relieve iron starvation of a heme oxygenase mutant; however, this contaminating iron would not be expected to affect hemin-mediated toxicity. Alternatively, the growth of the isdG mutant strain between 1 to 5 μM hemin could be due to the presence of additional as-yet-unidentified heme-degrading enzymes in B. anthracis.
To expand on the observation that inactivation of isdG increases the sensitivity of B. anthracis to hemin, a hemin toxicity assay was developed. In this assay, exposure of B. anthracis to concentrations of hemin above 1 μM led to hemin-mediated toxicity after 1 h (Fig. 6). This toxicity is concentration dependent and becomes more pronounced over time, with concentrations of hemin above 30 μM eliminating detectable B. anthracis strain Sterne after 2 h. Inactivation of isdG significantly increases hemin-mediated toxicity to the point where, after 1 h, we were unable to detect live bacteria upon exposure of B. anthracis isdG::ermC to concentrations of hemin above 10 μM (Fig. 6). These results demonstrate that IsdG not only degrades hemin to release free iron as a nutrient source but also protects the bacterium against the damaging effects caused by the accumulation of intracellular hemin.
The observation that isdG is required to protect against hemin-mediated toxicity suggests that IsdG is involved in protecting B. anthracis against reactive oxygen species generated upon exposure to high amounts of hemin. If this were the case, the hemin-mediated toxicity might be augmented by the respiratory burst of activated macrophages. To test this hypothesis, wild-type and isdG::ermC strains were measured for their ability to survive and grow in activated macrophages. We did not identify differences in the growth rate of wild-type and isdG::ermC strains in activated macrophages (data not shown). These results demonstrate that hemin degradation does not play a role in growth inside activated macrophages, suggesting that hemin degradation is important at sites of infection outside the macrophage-associated replication cycle of B. anthracis.
Role of IsdG in virulence during A/J mouse infections. B. anthracis secretes lethal toxin and edema toxin to cause anthrax disease (11). Three pXO1 virulence plasmid genes encode subunits for both toxins, pag (protective antigen [PA]), lef (lethal factor), and cya (edema factor), as PA performs binding and host cell transport functions for both lethal factor and edema factor (11). The toxin genes are known to be essential for disease progression in multiple animal models of anthrax cutaneous infection, including guinea pigs and A/J mice (5, 6, 34). Further, antibodies against PA appear to be a critical factor in protective immunity (26, 50). B. anthracis strain Sterne lacks the pXO2 virulence plasmid, encoding the capABCD genes responsible for synthesis of the poly-D-glutamic acid capsule (17, 33, 46). The glutamate capsule of B. anthracis is essential for the pathogenesis of cutaneous anthrax infections in mice and, presumably, in many other animal infections (15); however, strain Sterne retains significant virulence in the A/J mouse model, as these animals display significant defects in phagocytic killing of bacterial pathogens (51, 52).
B. anthracis strain Ames 50% lethal doses of 50 spores are required for the development of lethal anthrax disease in mice (34), whereas a subcutaneous 50% lethal dose of 106 Sterne spores is required to generate a similar disease (34). Welkos and colleagues showed that subcutaneous infection of A/J mice with B. anthracis strain Sterne spores leads to an acute lethal disease at a dose of 102 to 103 spores (51). Subcutaneous infection of A/J mice with 60,000 spores of either B. anthracis strain Sterne or isdG::ermC killed 100% of the animals within 3 days of infection. In addition, low-dose infection with 600 spores of either B. anthracis strain Sterne or isdG::ermC caused a lethal infection in 40 to 50% of the animals by 5 days postinfection (Fig. 7). These results suggest that IsdG-mediated heme degradation is not required for the ability of B. anthracis strain Sterne to cause systemic anthrax in the A/J mouse model of infection.
DISCUSSION
As observed for most bacterial pathogens, B. anthracis requires iron to multiply and to initiate and maintain pathogenic processes that lead to anthrax disease. The complex nature of the B. anthracis life cycle, combined with multiple potential entry points into the host, presumably necessitates diverse iron acquisition systems in this organism. Early in infection, the anthrax spore is presumed to be engulfed by circulating macrophages. After engulfment, B. anthracis spores germinate inside the macrophage, requiring iron for the vegetative cells to multiple and divide (13, 22, 23). Heme-containing proteins inside the macrophage are not predicted to be in abundance, and presumably reside primarily in host membranes, making them unlikely iron sources for intracellular bacilli. Due to the unavailability of intracellular heme, it seems more likely that the labile iron pool (8) or iron complexed to iron mobilization or storage proteins such as transferrin or ferritin is the primary iron source utilized during the intracellular life cycle of B. anthracis. These host iron sources are believed to be the primary target of bacterial siderophores, potentially explaining the severe decrease in virulence exhibited by a B. anthracis siderophore synthesis mutant (9). Once B. anthracis lyses the macrophage and enters the host bloodstream, additional iron sources are likely required for infection to progress. The most abundant iron source in the blood of mammalian hosts is in the form of hemoproteins such as hemoglobin (12). To liberate and utilize hemoglobin as an iron source, bacterial pathogens must have a mechanism capable of lysing the host's red blood cells. In the case of B. anthracis, this lysis might be mediated by cytolysins such as the cholesterol-dependent anthrolysin O (41). Once hemoglobin is liberated from lysed erythrocytes, systems responsible for hemoglobin binding, heme removal and transport into the cytoplasm, and heme degradation may be required for the utilization of heme as an iron source.
The isd system has been shown to be responsible for a portion of the binding and transport of heme iron into the cytoplasm of S. aureus (30). Homologous systems have been identified in numerous gram-positive pathogens, including Clostridium tetani, Listeria monocytogenes, and B. anthracis, implying that a conserved mechanism of heme binding, transport, and utilization exists in this group of human pathogens (45). As has been shown in S. aureus, B. anthracis presumably utilizes the Isd system to recognize host hemoproteins, transport heme into the bacterial cytoplasm, and degrade heme to release free iron. Two proteins of the staphylococcal Isd system, IsdG and IsdI, are monooxygenases responsible for the degradation of heme (43). IsdG and IsdI are members of the IsdG family of heme monooxygenases characterized by a -barrel type fold, assembled from the IsdG homodimer with two catalytic sites defined by the conserved NWH triad (55). Initial biochemical and structural characterization of the staphylococcal members of the IsdG family have been performed; however, the role of IsdG family-mediated heme degradation in the biology and virulence of a gram-positive pathogen has not been reported.
Here we show that B. anthracis expresses an IsdG family member possessing the conserved NWH triad that is also involved in hemin degradation. B. anthracis IsdG degrades hemin in vitro and in vivo to release free iron. The degradation product of BaIsdG-mediated hemin degradation has an HPLC elution profile similar to that of the degradation products produced upon SaIsdG- and SaIsdI-mediated hemin degradation, implying that all of these enzymes catalyze the degradation of hemin to release free iron and form bilverdin (43). Inactivation of B. anthracis isdG leads to an inability to utilize low amounts of hemin as a sole iron source and also prevents the ability of B. anthracis to grow in the presence of high levels of hemin (Fig. 5 and 6). This heme-mediated toxicity suggests that not only is IsdG-mediated heme degradation necessary for the utilization of hemin as an iron source, it is also involved in protection against the toxic effects of heme caused via the buildup of oxygen radicals. Ascribing a protective function to a heme oxygenase is not without precedence, as a primary function of vertebrate heme oxygenases is protection of the host against the toxic effects of heme (10, 24) and an effect of heme oxygenases on heme-mediated toxicity has been reported in the genus Neisseria (59). Inactivation of B. anthracis isdG did not affect the ability of the organism to proliferate inside the macrophage cell line J774, suggesting that heme degradation may not play a role in macrophage replication of B. anthracis. However, inactivation of srtB decreases growth of B. anthracis in the mouse macrophage-like cell line J774A.1 (60), suggesting that a functional heme uptake system is required for survival in this host niche. It is possible that the loss of IsdG is compensated for by additional enzymes in B. anthracis that are capable of degrading heme. Further studies are needed to delineate between the requirement for IsdG-mediated heme degradation in acquisition of iron as a nutrient and protection against heme-mediated toxicity.
Inactivation of isdG did not decrease the ability of B. anthracis strain Sterne to mediate disease in the A/J mouse model of systemic infection. A number of possible mechanisms explain this lack of an effect on virulence. Based on a strict requirement for siderophore-acquired iron in B. anthracis pathogenesis (9), it is possible that the infecting bacillus satisfies its iron requirement through siderophore-mediated acquisition of nonheme iron. This would imply that heme iron acquisition is not a prominent iron acquisition strategy during the pathogenesis of anthrax. Alternatively, the Sterne-A/J infection model might not adequately represent the infectious process inside a human. This possibility is supported by the absence of the pXO-2 virulence plasmid in B. anthracis strain Sterne and the immune deficiency of the A/J mouse strain (15, 51). We favor the hypothesis that additional heme-degrading enzymes exist in B. anthracis that have yet to be identified. This supposition is based largely on the measurable growth of the isdG::ermC strain at hemin concentrations between 1 and 5 μM. Multiple heme-degrading enzymes within a single organism have been reported for Pseudomonas aeruginosa (49) and S. aureus (43), and a recent genomic analysis of bacterial heme oxygenases proposed the existence of distinct heme-degrading enzyme families in bacteria (18). Using genomic analyses, we have been unable to identify any additional candidate heme oxygenases in the genome of B. anthracis, including those of the HO family (hmuO [38], pigA [35], hemO [59]) or the IsdG family (isdG, isdI [43]). In addition, we have not identified any potential members of the hutZ/shuS heme iron utilization genes (56, 57) in the B. anthracis genome. Together, these observations support the possibility that members of an additional, as yet unidentified heme-degrading enzyme family exist in B. anthracis.
Based on sequence analysis and the results shown here, we hypothesize that heme iron is acquired through the B. anthracis Isd system. In our model, hemoproteins are recognized by the putative cell wall-anchored proteins IsdA and IsdC and heme is removed and transported through the IsdEFX membrane transport system. Once inside the cytoplasm, heme is degraded by IsdG acting as a heme monooxygenase to release free iron and form biliverdin. IsdG-mediated heme degradation can provide nutrient iron to the rapidly multiplying bacillus as well as protect B. anthracis from heme-mediated toxicity that might be encountered upon exposure to oxidative radicals. A lack of a role for IsdG in growth inside an activated macrophage, combined with a requirement for IsdG to protect against heme-mediated toxicity, suggests that this activity might be important for protecting the bacterium against the large amounts of endogenous reactive oxygen species that would be formed during the cellular metabolism associated with the rapid microbial expansion that is characteristic of anthrax (29).
The demonstrated importance of heme iron during infection of other gram-positive pathogens (44), combined with the lack of a role for IsdG in the pathogenesis of B. anthracis reported here, supports the notion that additional heme-degrading enzymes exist in this organism. Recent advances in the availability of B. anthracis genome sequences (36), combined with an increased appreciation for the role of bacterial heme oxygenases in iron acquisition (18), will contribute greatly to our ability to further investigate the possibility that B. anthracis encodes multiple heme-degrading enzymes.
ACKNOWLEDGMENTS
We thank members of the Schneewind laboratory for critical reading of the manuscript as well as Joshua M. Barnett, Christina Tam, Luciano Marraffini, and Kristin DeBord for experimental assistance in the course of these studies.
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