Utilization of Host Iron Sources by Corynebacterium diphtheriae: Multiple Hemoglobin-Binding Proteins Are Essential for the Use of Iron from the Hemoglobin-Haptoglobin Complex

Abstract

The use of hemin iron by Corynebacterium diphtheriae requires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized by C. diphtheriae. We show that several C. diphtheriae strains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that an htaA deletion mutant of C. diphtheriae strain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that a chtA-chtC double mutant is also unable to use Hb-Hp iron. Single-deletion mutants of chtA or chtC use Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding. C. diphtheriae was also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source by C. diphtheriae requires multiple iron-regulated surface components.

INTRODUCTION

Corynebacterium diphtheriae is a Gram-positive bacterial pathogen that is associated with infections of the upper respiratory tract or skin in humans. In respiratory diphtheria, C. diphtheriae colonizes the nasopharynx or adjacent regions, where it produces the iron- and DtxR-regulated diphtheria toxin, an exotoxin that is responsible for the severe symptoms associated with infection by this organism (1, 2). Relatively few studies have identified factors that are critical for the survival and adherence of C. diphtheriae in the human host. Recent in vitro studies reported that specific pilus structures are associated with adherence of C. diphtheriae to various epithelial cells (3), and factors involved in iron acquisition are essential for the growth of C. diphtheriae under iron-depleted conditions (4, 5). Numerous factors in bacteria that are expressed under low-iron conditions are involved in the transport and metabolism of iron, and many bacterial pathogens encode systems that facilitate the utilization of host iron sources, including transferrin, lactoferrin, and various heme proteins such as hemoglobin (Hb), hemopexin (Hpx), human serum albumin (HSA), and myoglobin (Mb) (6–8). Another significant source of host iron is the Hb-haptoglobin (Hp) complex. The release of Hb into serum following cell lysis results in the formation of Hb dimers that are quickly bound by Hp, forming a tightly bound Hb-Hp complex (9). The Hb-Hp complex has been proposed to be a major source of host iron for certain bacterial pathogens (10). While the importance of systems involved in iron acquisition in C. diphtheriae is not known, the region colonized by C. diphtheriae is likely a low-iron environment (diphtheria toxin is expressed only under iron-limited conditions), which suggests that iron acquisition is essential for survival in the host.

In Gram-negative bacteria, the use of hemin or Hb iron initially requires binding of the heme source to outer membrane receptors and the subsequent uptake of hemin into the periplasmic space. Hemin-specific substrate binding proteins in the periplasm bind hemin and direct the porphyrin to hemin-specific ABC transporters for passage through the cytoplasmic membrane (11, 12). Gram-negative and Gram-positive bacteria both use ABC-type hemin transporters to mobilize hemin through the cytoplasmic membrane; however, the binding of hemin or heme proteins at the surface of these bacteria is quite distinct, in that many Gram-positive bacteria utilize cell wall- or membrane-anchored proteins to initially bind hemin or heme proteins. These surface proteins are designated iron-regulated surface determinants (Isd) in Staphylococcus aureus and Bacillus anthracis (13, 14), while in Streptococcus pyogenes, the Shr and Shp proteins are the primary surface-exposed hemin- or Hb-binding proteins (15, 16). These various surface-anchored proteins in Gram-positive bacteria specifically bind either hemin or Hb. Following binding, the hemin is mobilized by a series of proteins through the cell wall, where hemin is ultimately transferred to a hemin-specific ABC transporter. The region required for binding hemin or Hb in this group of Gram-positive bacterial proteins is an approximately 125-amino-acid region termed a NEAT domain that is specific for either hemin or Hb (17, 18).

Systems involved in iron acquisition in C. diphtheriae include a siderophore and its cognate transport proteins (4), as well as a hemin iron acquisition system encoded by genes in the hmu gene cluster (5). The hmu region contains genes for the ABC-type hemin transporter, HmuTUV, and two hemin-binding surface-anchored proteins, HtaA and HtaB (5). HtaA is a 61-kDa membrane-anchored protein that contains two surface-exposed conserved regions (designated CR1 and CR2) that bind hemin and Hb (19). C. diphtheriae htaA null mutants exhibit a decreased ability to use hemin and Hb as sole iron sources, suggesting a direct role for HtaA in the use of hemin iron. Site-directed mutagenesis studies identified two conserved tyrosine residues and a conserved histidine residue in the CR domains that were critical for hemin and Hb binding by HtaA, and the most conserved tyrosine residue, CR2-Y361, was essential for the hemin utilization function of HtaA (19). No significant sequence similarity exists between the hemin or Hb-binding NEAT domains and the CR domains in Corynebacterium species; however, the binding of hemin appears to involve conserved tyrosine residues in both regions (18, 19).

The hemin-binding HtaB protein (36 kDa) contains a single CR domain that is essential for hemin binding but is not involved in Hb binding (19). Recent studies with C. diphtheriae htaB mutants suggest that HtaB is involved in hemin uptake, and in vitro experiments showed that HtaB acquires hemin from HtaA (19, 20). Taken together, these findings suggest that HtaB likely functions as an intermediate in the transport of hemin through the cell wall.

In a previous report, we described two unique genetic regions, designated chtA-chtB and cirA-chtC, that encode hemin- and Hb-binding surface proteins with sequence similarity to HtaA and HtaB. Both of these genetic systems are organized in two-gene operons, and their expression is repressed by DtxR in an iron-dependent manner (20). The chtA-chtB operon is flanked by two identical insertion sequences, and the overall genetic arrangement of this region suggests that chtA-chtB is part of a composite transposon. The cirA-chtC region is not associated with any putative mobile genetic elements and is not closely linked to the chtA-chtB operon. ChtA (83.9 kDa) and ChtC (74.3 kDa) each contain a single N-terminal CR domain, and both possess a C-terminal transmembrane region which is believed to tether these proteins to the cytoplasmic membrane in a manner similar to that of HtaA and HtaB. While ChtA and ChtC exhibit significant sequence similarity to each other over the full length of the protein, they show similarity to HtaA primarily within the CR domains. The ChtB (32.6-kDa) protein shows high sequence similarity to HtaB (44% identity and 63% similarity), and recent studies suggest that the two proteins have similar heme transport functions (20). Both ChtA and ChtC are able to bind hemin and Hb in vitro, and the ChtA CR domain is required for this activity; a function for the 450-amino-acid C-terminal region for ChtA and ChtC was not determined. Mutant strains of C. diphtheriae 1737 carrying nonpolar deletions in either chtA or chtC showed no defect in the ability to use either hemin or Hb as an iron source, and a biological function for ChtA or ChtC was not determined in that study (20).

In this study, we assessed the ability of C. diphtheriae to utilize a variety of heme-containing proteins as iron sources, including the Hb-Hp complex, HSA, Mb, and Hpx. We show that C. diphtheriae is able to use all of these hemoproteins, with the exception of hemopexin. Studies with C. diphtheriae mutants indicate that HtaA and either ChtA or ChtC are essential for the use of the Hb-Hp complex as an iron source. The findings in this study, for the first time, show a function for the ChtA and ChtC proteins and show that two surface-exposed proteins are required for the use of the Hb-Hp complex as an iron source.

MATERIALS AND METHODS

Bacterial strains and media.

Escherichia coli and C. diphtheriae strains used in this study are listed in Table 1. Luria-Bertani (LB) medium was used for culturing of E. coli, and heart infusion broth (Difco, Detroit, MI) containing 0.2% Tween 80 (HIBTW) was used for routine growth of C. diphtheriae strains. Bacterial stocks were maintained in 20% glycerol at −80°C. Antibiotics were added to LB medium at 50 μg/ml for kanamycin and 100 μg/ml for ampicillin and to HIBTW for C. diphtheriae cultures at 50 μg/ml for kanamycin. Modified PGT (mPGT) is a semidefined low-iron medium that has been previously described (21). Antibiotics, ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA), Tween 80, Hp (human; Hp1-1), HSA, and Mb (human) were obtained from Sigma Chemical Co. Purified Hb (human) was purchased from MP BioMedical, and Hpx (human) was from Cell Sciences.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid	Relevant characteristics or use	Reference or source
C. diphtheriae strains
1737	Wild type, Gravis biotype, tox+	24
1737htaAΔ	htaA deletion mutant of 1737	5
1737hmuTUVΔ	hmuTUV deletion mutant of 1737	5
1737hmuΔ	hmu operon deletion mutant of 1737	5
1737chtAΔ	chtA deletion mutant of 1737	20
1737chtCΔ	chtC deletion mutant of 1737	20
1737chtAΔ/chtCΔ/htaAΔ	Deletion of chtA, chtC, and htaA in 1737	20
1737chtAΔ/chtCΔ	Deletion of chtA and chtC in 1737	20
C7 (−)	Wild type, tox negative	36
1716	Wild type, Gravis biotype, tox+	24
1718	Wild type, Gravis biotype, tox+	24
G4193	Wild type, tox negative	24
PW8	Wild type, tox+ U.S. vaccine strain	37
E. coli strains
BL21(DE3)	Protein expression	Novagen
DH5α	Cloning strain	Invitrogen
XL1-Gold	Mutagenesis strain	Stratagene
Plasmids
pGEX-6P-1	Expression vector (GST fusion); Ampr	GE Healthcare
pET24(a)+	Expression vector; Knr	Millipore
pKN2.6Z	C. diphtheriae shuttle vector; Knr	23
pKN-htaA	pKN2.6Z carrying the htaA gene	5
pKNQ-cys	pKN-htaA, Cys to Alaa	This study
pKN-htaA361	pKN-htaA with Y361A mutation	19

^aThe four cysteine residues in HtaA were changed to alanine.

Plasmid construction.

Plasmids used in this study are listed in Table 1. The vector pGEX-6P-1 (Amersham) was used for the expression of N-terminal glutathione S-transferase (GST)-tagged HtaA-wt, HtaA-Y49A, HtaA-Y361A, HtaA-CR2, HtaA-CR2Y361A, and HtaA-CR1. The construction of these plasmids has been previously described (5, 19). The N-terminally Strep-tagged proteins HtaA, HtaA-CR2, ChtA, ChtA-CR, ChtA-C-terminal, and ChtC were constructed using the pET24a vector (Novagen) and have been previously described (19, 20). Q-Cys, which was constructed from the Strep-tagged HtaA protein, contains Cys-Ala substitutions at all four cysteine residues.

Mutant construction.

The construction of nonpolar deletion mutations in C. diphtheriae 1737 has been previously described (5, 20). Site-directed mutations were made using the QuikChange Lightning kit (Stratagene) according to the manufacturer's instructions. Briefly, 125 ng of each primer containing the targeted base change and 50 ng of plasmid template were used in the QuikChange reaction. Methylated template DNA was removed from the reaction by digestion with DpnI restriction endonuclease, and mutagenized DNA was recovered by transformation into XL1-Gold-competent cells. The presence of the base changes was confirmed by sequence analysis. Plasmids used for site-directed mutagenesis were pKN-htaA and pET24a carrying the cloned htaA gene.

Protein expression.

Recombinant proteins were expressed in BL21(DE3) carrying various cloned genes. Strains carrying expression plasmids were grown in 100 ml of LB medium at 37°C to mid-log phase, at which time 1 to 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added and the cultures were allowed to grow for additional 2 to 3 h at 16 to 37°C before harvesting. The cultures were washed in 10 ml of 20 mM Tris-HCl (pH 7.5), and the pellet was either stored at −20°C until use or resuspended in lysis buffer for lysis by French pressure cell or FastPrep lysis. Cell debris was removed by centrifugation at 12,000 × g at 4°C, and the supernatant fraction containing the soluble protein was purified on Strep-Tactin columns (Neuromics) for Strep-tagged constructs. Soluble lysates containing the GST-tagged constructs were purified using GST-resin (GE Healthcare) by a batch method according to the manufacturer's instructions.

Native PAGE gels.

Hb and Hp were mixed at a 1:1 molar ratio and incubated at room temperature for 45 min, with mixing every 15 min. Native sample buffer (without SDS; Bio-Rad) was added, and the samples were run on a 10% native gel without SDS with 1× Tris-glycine buffer (Bio-Rad) for 1 h at 200 V, followed by staining with Bio-Safe Coomassie (Bio-Rad) according to the manufacturer's instructions.

Heme protein iron utilization assays.

The hemoglobin utilization assay has been described previously (5). Briefly, C. diphtheriae strains were grown overnight (20 to 22 h at 37°C) in HIBTW. The next day, overnight cultures were diluted with 1:2 with HIBTW and incubated for 1 h at 37°C. Then, 500-μl volumes of the cultures were centrifuged and resuspended in 1 ml of mPGT medium with 1 μM FeSO₄. Strains were grown for several hours at 37°C until log phase, at which time bacteria were inoculated at an optical density at 600 nm (OD₆₀₀) of 0.03 into fresh mPGT medium that contained Hb at 25 μg/ml and the iron chelator EDDA at 10 μM to scavenge free iron. After 20 to 22 h of growth at 37°C, the OD₆₀₀ of the cultures was determined. All strains grew well in the presence of 1 μM FeSO₄, and all strains were inhibited for growth under low-iron conditions (10 μM EDDA). The same procedure was followed for growth in the presence of Hb-Hp, heme-HSA, Mb, and heme-Hpx. For growth in the presence of Hb-Hp, Hb and Hp were mixed at a 1:1 molar ratio (Hb, 25 μg/ml, and Hp, 35 μg/ml) and then incubated at room temperature for 45 min, with mixing every 15 min, before addition to overnight cultures. To produce heme-HSA, HSA was bound to hemin at a 1:1 molar ratio, incubated at 37°C for 1 h, and dialyzed twice in phosphate-buffered saline (PBS) plus glycerol to remove free hemin. Heme-HSA was examined by UV-visual spectroscopy to confirm hemin binding prior to use in the growth assays. The same procedure was followed for heme-Hpx, but with incubation on ice for 20 min instead of 37°C for 1 h. Statistical analysis to assess differences in growth was done with GraphPad Prism.

Protein binding studies.

An enzyme-linked immunosorbent assay (ELISA) method was used to assess the abilities of various test proteins to bind immobilized Hb, Hb-Hp, heme-HSA, and Mb. Microtiter plates (96-well polystyrene plates; Costar) were coated overnight at 37°C with 50 μl of Hb (25 μg/ml), Hb-Hp (25 μg/ml and 35 μg/ml, respectively), heme-HSA (25 μg/ml), or Mb (25 μg/ml) in PBS. After incubation, the plates were washed with PBST (PBS plus 0.05% Tween 20) and blocked for 1 h with 5% Blotto in PBST, followed by incubation with the test protein at various protein concentrations (50 μl) for 1 h. After incubation, the plates were washed with PBST, and primary antibodies, anti-GST (1:1,000; GE Healthcare) or anti-Strep (1:1,000; Abcam Inc.), were then added to the plates for 1 h, followed by an additional wash and then a 1-h incubation with appropriate alkaline phosphatase-labeled secondary antibodies. All incubations were performed at 37°C. The plates were developed in the dark with 50 μl of p-nitrophenylphosphate (pNPP; Sigma) at room temperature. A final absorbance reading was taken when the positive-control sample (HtaA-wt) achieved an OD₄₀₅ of approximately 1. The concentration of test protein required to achieve half-maximum binding (EC₅₀) was determined using the nonlinear regression model (saturation binding kinetics) from software available in GraphPad Prism.

RESULTS

C. diphtheriae utilizes the Hb-Hp complex as an iron source.

Previous studies showed that C. diphtheriae uses hemin and Hb as sole iron sources during growth in iron-depleted medium (22, 23). To investigate whether C. diphtheriae is able to use iron from other relevant heme-containing proteins, we assessed the abilities of six different C. diphtheriae strains to use the Hb-Hp complex as an iron source. Bacterial strains were examined for the ability to grow in low-iron mPGT medium in which either Hb or the Hb-Hp complex (at a 1:1 molar ratio) (Fig. 1A) was supplied as the only iron source. All strains tested were able to utilize iron from both Hb and Hb-Hp, although a preference for Hb was observed in the six strains examined (Fig. 1B). Hp did not enhance growth for any of the strains (Fig. 1B). Strains 1737, 1716, and 1718 are members of a related clonal group that dominated the diphtheria outbreak in the former Soviet Union (FSU) in the 1990s (24). Despite the similarities of these strains, they exhibited different levels of growth enhancement with Hb and Hb-Hp. Strains 1716 and 1718 showed markedly reduced growth with Hb relative to that of all other strains. The reason for this reduced growth with Hb is unclear, since these three strains from the diphtheria outbreak in the FSU contain all of the known genes involved in either hemin transport or hemin binding, including the hmu operon and the chtAC and cirA-chtC genes (20). Strains C7, G4193, and PW8 are not closely related and carry deletions of chtA and chtC, while strains C7 and PW8 also contain point mutations in htaA (20, 25). All three of these strains showed strong growth stimulation by Hb, while growth enhancement with the Hb-Hp complex was 3-fold lower than that seen with Hb (Fig. 1B). The difference in growth enhancement between Hb and Hb-Hp with the FSU strains was significantly less and ranged from 5% to 25%.

FIG 1.

(A) Native gel containing Hb, Hp, and Hb-Hp complex (at a 1:1 molar ratio). (B) Growth of various C. diphtheriae strains in low-iron mPGT media containing Hb, Hb-Hp, or Hp as the sole iron source. All strains exhibited similar levels of growth with Hp; only the Hp result for 1737 is shown (Hp was used at 35 μg/ml). Growth with Hp is significantly reduced relative to growth with Hb-Hp for all strains. Values represent the means from three independent experiments (±SD).

HtaA is essential for the use of the Hb-Hp complex as an iron source.

The hmu operon encodes proteins that are important for the use of hemin and Hb as iron sources, including the HmuTUV-ABC transporter and the surface proteins HtaA and HtaB (Fig. 2A). C. diphtheriae strain 1737 carrying a deletion of the htaA gene shows significantly reduced growth in low-iron medium when Hb is the sole iron source (5) (Fig. 2B). Growth assay results shown in Fig. 2B reveal that strain 1737 carrying a nonpolar deletion in htaA is unable to grow when Hb-Hp is the sole iron source, indicating that HtaA is essential for the use of Hb-Hp iron. Deletions of the hmuTUV genes or the hmu operon also abolished growth in the presence of Hb-Hp (Fig. 2B). The presence of the cloned wild-type (wt) htaA gene (pKN-htaA) in 1737htaAΔ restored growth of the mutant in the presence of Hb-Hp (Fig. 2C); however, a point mutation at Y361, a tyrosine residue important for hemin and Hb binding by HtaA (19), was not able to restore growth in the presence of Hb-Hp (Fig. 2C).

FIG 2.

(A) Genetic map of the hmu gene cluster. Arrows indicate direction of transcription. CR domains for HtaA and HtaB are shown. Below the map are shown the regions deleted in the various nonpolar deletion mutants constructed from strain 1737. (B) Growth of strain 1737-wt and various nonpolar deletion mutants in low-iron media containing either Hb or Hb-Hp. The asterisk indicates that growth with Hb-Hp is significantly different from that of the wild type (P < 0.0001). (C) Growth of strain 1737htaAΔ carrying various pKN-2.6 plasmids. Growth conditions are the same as described for panel B. The asterisk indicates that growth with Hb-Hp is significantly different from that with the pKN2.6-vector (P < 0.0001). Values shown for panels B and C represent the means from three independent experiments (±SD).

HtaA binds the Hb-Hp complex.

HtaA and various derivatives were examined for the ability to bind the Hb-Hp complex using an in vitro ELISA procedure. Full-length HtaA and the CR2 domain showed the strongest binding to Hb-Hp (HtaA EC₅₀, 210 nM; CR2 EC₅₀, 200 nM), while the CR1 domain exhibited about 2-fold-weaker binding (CR1 EC₅₀, 370 nM) (Fig. 3). The Y361A derivatives of full-length HtaA (EC₅₀, 870 nM) and CR2 (no binding detected) showed a marked reduction in binding to Hb-Hp, indicating the importance of this critical tyrosine in the interaction with heme-containing proteins. The Y49A derivative, which alters the homologous tyrosine in the CR1 domain, showed only a slight effect on Hb-Hp binding (HtaA-Y49A EC₅₀, 270 nM). A quantitative comparison for the binding of all proteins to Hb-Hp assessed at a 200 nM concentration (EC₅₀ for the CR2 domain) is presented in Fig. 3B. HtaA showed background levels of binding to Hp (Fig. 3B), suggesting that most, if not all, of the interaction with the Hb-Hp complex is through the Hb moiety.

FIG 3.

(A) HtaA and derivatives were assessed for the ability to bind Hb-Hp by ELISA. GST was included as a negative control. Experiments were repeated at least three times, with similar results. Results of a representative experiment are shown. (B) Binding of HtaA and various derivatives to Hb-Hp at a 200 nM concentration is shown. Binding of HtaA-Y361A and CR2-Y361A to Hb-Hp was significantly different from binding with HtaA-wt and CR2, respectively (**, P < 0.0001). Binding of CR1 to Hb-Hp was significantly different from that with HtaA-wt (***, P < 0.05). *, HtaA does not bind Hp (HtaA-Hp). Values represent the means from three independent experiments (±SD).

ChtA or ChtC is required for the use of iron from the Hb-Hp complex.

The ChtA and ChtC proteins are encoded on unlinked two-gene operons that are regulated at the transcriptional level by iron (Fig. 4A). We previously showed that the surface-anchored ChtA and ChtC proteins bind hemin and Hb but are not required for the use of these compounds as iron sources (20). A function for these proteins was not demonstrated in that earlier study. To determine whether these proteins are required for the utilization of iron from Hb-Hp, we tested mutants of strain 1737 that had deletions of chtA, chtC, and both chtA and chtC. Mutants with single deletions of chtA or chtC were able to use Hb and Hb-Hp as iron sources in a manner similar to that of the wild-type strain; however, the double mutant (chtA chtC) was fully abolished in its ability to use Hb-Hp iron (Fig. 4B). A triple mutant of strain 1737 that had deletions of htaA, chtA, and chtC was also unable to use Hb-Hp iron and was further diminished for growth with Hb, consistent with the phenotype for an htaA mutant (Fig. 4B). These findings indicate that both HtaA and either ChtA or ChtC are essential for the use of the Hb-Hp complex as an iron source in C. diphtheriae. ChtA and ChtC exhibit sequence and structural similarities, and the results in this growth assay suggest that the proteins can substitute for one another in the use of Hb-Hp iron.

FIG 4.

(A) Genetic map of the chtA-chtB and cirA-chtC regions. “P” (and arrow) indicates the direction of transcription from DtxR and iron-regulated promoters. “IS” indicates insertion sequences; “CR” indicates a conserved region containing hemin/Hb-binding domains. (B) Growth of 1737-wt and various nonpolar deletion mutants in low-iron media containing either Hb or Hb-Hp as the sole iron source. Values represent the means from three independent experiments (±SD). The asterisk indicates that growth with Hb-Hp is significantly different from that with the wild type (P < 0.0001).

Analysis of ChtA and ChtC binding to the Hb-Hp complex.

Previous studies showed that ChtA and ChtC bind hemin and Hb and that the N-terminal CR domain of ChtA is required for the binding activity (20) (Fig. 5A). To assess binding to Hb and to the Hb-Hp complex, purified recombinant proteins that contained N-terminal Strep tags were tested for in vitro binding using the previously described ELISA procedure. While ChtA and ChtC showed characteristics for binding to Hb similar to those observed with HtaA (Fig. 5A), binding to the Hb-Hp complex was markedly reduced with ChtA and ChtC relative to that observed with HtaA (Fig. 5B and C). The ChtA-CR domain showed enhanced binding to Hb-Hp relative to the full-length ChtA protein (ChtA-CR EC₅₀, 890 nM; ChtA EC₅₀, >2 μM); however, the ChtA-CR domain did not bind Hb-Hp with as high affinity as the HtaA-CR2 domain (EC₅₀, 380 nM) (Fig. 5B and C). The Strep-tagged HtaA proteins exhibited higher EC₅₀s than those observed with the GST-tagged proteins (Fig. 3B). The 450-amino-acid ChtA C-terminal region does not bind Hb, Hp, or the Hb-Hp complex, and a function for this region has not been determined (Fig. 5A and B and data not shown for Hp binding).

FIG 5.

(A) HtaA, ChtC, ChtA, and derivatives of ChtA were assessed for the ability to bind Hb by ELISA. Experiments were repeated at least three times, with similar results. Results of a representative experiment are shown. (B) The same proteins as in panel A were assessed for the ability to bind Hb-Hp by ELISA. Experiments were repeated at least three times, with similar results. Results of a representative experiment are shown. (C) Hb-Hp binding was assessed with proteins indicated in panel B at a 400 nM concentration. Values represent the means from three independent experiments (±SD). Binding of ChtA and ChtC to Hb-Hp was significantly different from that with HtaA (*, P < 0.001).

HtaA facilitates the utilization of iron from human serum albumin and myoglobin.

The use of heme-HSA, heme-Hpx, and Mb as iron sources has not been widely studied, although several bacterial pathogens are able to use these heme-containing proteins (26–29). HSA and hemopexin are the dominant serum proteins involved in the binding and subsequent removal of free heme from circulation, while Mb is structurally and functionally related to Hb and is present in muscle tissue. The release of Mb into circulation occurs after damage to muscle tissue, which may be caused by the action of diphtheria toxin at various host tissues.

The C. diphtheriae 1737 wild-type strain and various mutants were assessed for the ability to use heme-HSA, Mb, and heme-Hpx as sole iron sources during growth in iron-depleted medium. Growth assays showed that the wild-type strain was able to use both heme-HSA and Mb but not heme-Hpx as iron sources (Fig. 6A). Strain 1737 carrying a nonpolar deletion in either htaA or the hmuTUV genes exhibited significantly reduced levels of growth with heme-HSA and Mb relative to those of the wt strain (Fig. 6A), suggesting that the surface-anchored HtaA and the HmuTUV ABC transporter are required for wild-type levels of growth with these heme-containing proteins. The cloned htaA gene (pKN-htaA) was able to complement the 1737htaAΔ strain for growth with heme-HSA and Mb (Fig. 7B). Strain 1737 carrying mutations in chtA, chtC, or both genes (chtA chtC double mutant) used heme-HSA and Mb as iron sources in a manner similar to that of the wild-type strain (data not shown). These findings indicate that the HtaA protein is involved in the utilization of iron from a broad range of host heme-containing compounds.

FIG 6.

(A) Growth assay. C. diphtheriae 1737-wt and various nonpolar deletion mutants of 1737 were grown in low-iron medium containing heme-HSA, Mb, or heme-Hpx as the sole iron source. Values represent the means from three independent experiments (±SD). Asterisks indicate that growth is significantly different from that of the wild type (*, P < 0.0001; **, P < 0.0005). (B) An ELISA procedure was used to assess the binding of HtaA and associated proteins to heme-HSA. Experiments were repeated at least three times, with similar results. Results of a representative experiment are shown.

FIG 7.

(A) Structural map of HtaA. CR domains and flanking cysteine residues (C) are indicated. TM, transmembrane region; SP, signal peptide. (B) Growth of strain 1737htaAΔ carrying various pKN2.6 plasmids. Plasmid pKNQ-cys carries the cloned htaA gene that contains Cys-Ala substitutions at all four cysteine residues. Strains were grown in low-iron medium containing either heme-HSA, Hb-Hp, or Mb as the sole iron source. Values represent the means from three independent experiments (±SD). The asterisk indicates that growth is significantly different from that with the pKN2.6-vector (P < 0.005). (C) Purified HtaA and derivatives were assessed for the ability to bind Mb by ELISA. Q-Cys is purified HtaA with Cys-Ala substitutions at all four cysteine residues. Experiments were repeated at least three times, with similar results. Results of a representative experiment are shown. (D) Mb binding was assessed with proteins indicated in panel C at a 50 nM concentration. Values represent the means from three independent experiments (±SD). Binding of HtaA, CR2, and CR1 to Mb was significantly different from that of Q-Cys (*, P < 0.005).

HtaA binding studies with heme-HSA showed that the wild-type protein and the CR1 and CR2 domains are able to bind heme-HSA (Fig. 6B). The Y361A substitution in the full-length HtaA protein as well as in the CR2 domain (CR2-Y361A) resulted in reduced binding compared to that of the wild-type proteins.

Cysteine residues in HtaA affect Mb iron utilization and binding.

Analysis of the HtaA sequence revealed the presence of four cysteine residues that flank the CR1 and CR2 domains (Fig. 7A). No additional cysteine residues are present in the HtaA sequence. Cysteine residues are capable of forming disulfide bonds when present in surface-exposed proteins, which may result in unique structural modifications that impact function. To assess the role of these amino acids in HtaA activity, site-directed mutagenesis was used in the cloned htaA gene present on plasmid pKN-htaA to change all four cysteine residues to alanine; the new construct is designated pKNQ-cys. 1737htaAΔ/pKNQ-cys exhibited levels of growth comparable to those of 1737htaAΔ/pKN-htaA (wild-type htaA) when heme-HSA, Hb-Hp, and Hb were the sole iron sources (Fig. 7B and data not shown for Hb). However, plasmid pKNQ-cys was unable to complement the defect in 1737htaAΔ when Mb was used as the iron source (Fig. 7B). This finding suggests cysteine residues are required for the heme iron utilization function of HtaA but not for the other heme-containing proteins when Mb is provided as the sole iron source.

Binding studies showed that HtaA (EC₅₀, 60 nM) and the CR domains, CR1 (EC₅₀,190 nM) and CR2 (EC₅₀, 60 nM), exhibited significantly stronger binding to Mb than to any of the other previously examined heme-containing proteins (Fig. 7C and D). Surprisingly, the Q-cys variant of HtaA, which harbors the cysteine-to-alanine substitutions, showed an even higher affinity for Mb than the wild-type HtaA protein (EC₅₀, 20 nM) (Fig. 7C and D). Although the HtaA Q-cys protein shows stronger binding to Mb, the complementation study indicates that this HtaA derivative is unable to use Mb as an iron source. While it is possible that the stronger binding inhibits the release of heme from Mb, other conformational changes in HtaA caused by the loss of the cysteine residues may impact the use of Mb iron.

DISCUSSION

In respiratory diphtheria, the presence of a psuedomembrane, a necrotic and hemorrhagic lesion found in the nasopharyngeal region, is characteristic of severe disease caused by toxigenic strains of C. diphtheriae (30). The trauma associated with C. diphtheriae infection, including lesions caused by diphtheria toxin both at the local site of bacterial colonization and at distant sites in the host, likely results in an environmental milieu that may contain a variety of host iron sources, including heme-containing proteins such as Hb, Hb-Hp, HSA, Hpx, and Mb. Limited information is available regarding the various iron sources that are utilized by C. diphtheriae during infection. Most of what we know regarding iron source utilization by C. diphtheriae is derived from in vitro studies. Previous reports showed that C. diphtheriae produces a siderophore and its cognate transporter as well as a heme-specific uptake system during growth in low-iron medium (4, 5). Moreover, genomic analysis predicts that C. diphtheriae encodes several additional iron uptake systems (25). Many of the C. diphtheriae iron and heme transporters are predicted to be coordinately expressed with diphtheria toxin and therefore are likely produced during infection (4, 5, 25, 31). The abundance of putative iron transporters and the likelihood that C. diphtheriae colonizes a low-iron environment in the host suggest that the ability to acquire iron during infection is important for the virulence of C. diphtheriae.

The release of Hb into serum results in the rapid dissociation of the Hb tetramer into dimers (α₁β₁) which become irreversibly bound by the acute-phase protein Hp (9). Hp (phenotype, Hp 1-1) is found in mammalian serum as a α₂β₂ tetramer and is able to bind two α₁β₁ Hb dimers through its β-subunits (Fig. 8) (9). The binding of Hb to Hp is a strong noncovalent interaction, and the complex facilitates the clearance of Hb from circulation through binding to the CD163 macrophage receptor and subsequent uptake and processing by macrophages (32). Furthermore, an additional benefit of the Hb-Hp interaction is the protection of the host from oxidative damage caused by freely circulating Hb (33).

FIG 8.

Model of Hb-Hp-iron utilization in C. diphtheriae. The Hb-Hp complex is composed of a Hp tetramer in which each dimer binds an αβ dimer of Hb. HtaA in association with ChtA or ChtC is proposed to bind Hb-Hp at the cell surface by an unknown mechanism (?). Following binding, hemin is either extracted or naturally released from Hb-Hp and subsequently transported into the bacterium by a cascade process involving various surface proteins, including HtaA, HtaB, and HmuT. An ABC transporter, composed of HmuU and HmuV, mobilizes heme into the cytosol, where iron is released by the action of the heme oxygenase HmuO.

It was proposed that the Hb-Hp complex may be the most physiologically relevant host heme iron source available to certain invading bacterial pathogens (10). Several virulent bacterial species are known to utilize Hb-Hp iron, and certain bacterial surface proteins specifically bind the Hb-Hp complex. In Gram-negative bacteria, the Hb-Hp iron/heme utilization system is best characterized for Neisseria meningitidis and Haemophilus influenzae (10, 34). In N. meningitidis, two outer membrane proteins designated HpuB and HpuA are essential for the use of the Hb-Hp as an iron source. HpuB (88 kDa) is an iron-regulated TonB-dependent receptor that shares similarity to other outer membrane proteins involved in iron uptake, while HpuA (35 kDa) is a membrane-anchored lipoprotein that forms a complex with HpuB on the bacterial surface. The HpuAB complex is able to bind Hb, apo-Hp, and Hb-Hp, and both proteins are essential for the utilization of iron from Hb and the Hb-Hp complex (10). In H. influenzae, the ability to use the Hb-Hp complex as a heme source requires the products of the hgp genes, of which there exists anywhere from one to four copies in a given strain (34). The Hgp proteins in the well-studied strain H. influenzae HI1689 (HgpA, HgpB, and HgpC) are TonB-dependent outer membrane receptors that share a high level of sequence similarity and appear to have a redundant function with regard to the use of Hb-Hp. Mutations in all three genes are required to abolish the use of Hb-Hp as a heme source, although this triple mutant is still able to use Hb but at a reduced level (34).

Among Gram-positive bacteria, studies examining the use of Hb-Hp as an iron source have been described most extensively for S. aureus, in which the surface-anchored IsdH protein is involved in the binding to Hb-Hp, apo-Hp, and Hb. IsdH contains three NEAT domains, of which two bind Hb, Hp, and the Hb-Hp complex, while the third binds only hemin (18). Studies using an isdH mutant failed to demonstrate that IsdH is required for the use of Hb-Hp as an iron source in vitro (35), while in vivo studies suggest that IsdH is not essential for virulence in relevant animal models (8). The hemin- and Hb-binding protein IsdB from S. aureus shares significant sequence similarity to IsdH, and while IsdB is required for virulence and is essential for the use of Hb as an iron source, the ability of IsdB to bind or utilize the Hb-Hp complex as an iron source has not been reported.

In an earlier study, we were unable to identify a function for ChtA and ChtC (20). We show here that these proteins are involved in the use of the Hb-Hp complex as an iron source, and the findings suggest that ChtA and ChtC have redundant functions, since the deletion of both chtA and chtC is required for strain 1737 to lose the ability to acquire Hb-Hp iron. This study also revealed that HtaA was required for the use of Hb-Hp iron, suggesting that HtaA and either ChtA or ChtC are essential for the use of iron from Hb-Hp. While the mechanism by which these proteins utilize Hb-Hp iron is not known, bipartite systems are required for the use of Hb-Hp in pathogenic Neisseria species (see above). However, in Neisseria, the factors involved appear quite different from those in C. diphtheriae. The system in Neisseria is composed of two very distinct outer membrane proteins, while in C. diphtheriae, HtaA and ChtA/C share not only sequence similarity but also similarities in size, structure, cellular location, and the ability to bind hemin and Hb at CR domains, although they differ in their affinities for Hb-Hp, and they also differ in their abilities to use various iron sources. HtaA is involved in the use of free hemin, Hb, Hb-Hp, HSA, and Mb as iron sources, while ChtA and ChtC are involved only in the use of Hb-Hp iron, which suggests that the function of ChtA/C is more limited and more specific than that of HtaA. The CR domains of ChtC and ChtA show much greater similarity to each other than they do to the CR domains in HtaA (20); these sequence differences may be responsible for their differences in binding affinity toward Hb-Hp. ChtA and ChtC also contain a unique 450-amino-acid C-terminal region of unknown function. While this region is likely not involved in binding Hb, apo-Hp, or Hb-Hp, a structural role for this region in the use of Hb-Hp iron cannot be ruled out. It was observed that binding of the ChtA CR domain in the absence of the C-terminal region (ChtA-CR) resulted in enhanced binding to the Hb-Hp complex relative to that of the full-length ChtA protein (Fig. 5B and C), suggesting that, at least in vitro, the presence of the C-terminal region causes a reduction in binding to the Hb-Hp complex. Since both HtaA and ChtA/C are presumably positioned in a similar manner on the surface of the bacteria, it is possible that HtaA forms a complex with ChtA or ChtC to facilitate the binding and utilization of heme-iron from the Hb-Hp complex.

The results of the Hb-Hp binding study in Fig. 3 suggest that the CR2 domain is the dominant binding region in the full-length HtaA protein, although CR1 also contributes to binding. The contribution of the CR1 and CR2 domains in the binding to Hb-Hp at the surface of the bacterium is difficult to discern using in vitro methods, and it appears likely that the interaction of HtaA and ChtA/C with Hb-Hp in vivo is complex and will require additional in vitro and in vivo studies to better understand the binding interactions and the mechanism by which hemin is obtained from Hb-Hp and subsequently transported through the bacterial cell wall. A model depicting the utilization of iron from Hb-Hp is presented in Fig. 8.

In this study, we showed that C. diphtheriae is able to utilize a variety of host heme-containing proteins as iron sources, including Hb, Hb-Hp, heme-HSA, and Mb. We show in Fig. 1 that six strains, three of which are from the diphtheria epidemic in the FSU (1737, 1716, and 1718) and three of which are unrelated, were all able to use iron from both Hb and the Hb-Hp complex. Surprisingly, the three unrelated strains, C7, G4193, and PW8, lack the genes for both chtA and chtC and two of the strains (C7 and PW8) carry a defective htaA gene (the status of htaA in G4193 is not known) (20, 25). All of the FSU strains carry the chtA, chtC, and htaA genes (20). The findings suggest that the use of Hb-Hp iron is widespread among C. diphtheriae isolates but that some strains, such as C7, PW8, and G4193, use an alternative mechanism(s) that does not involve HtaA, ChtA, or ChtC. Further analysis of these strains is needed to understand the diversity of mechanisms involved in heme iron utilization in C. diphtheriae strains.