A growing body of evidence suggests that surface or secreted proteins with NEAr Transporter (NEAT) domains play a central role in haem acquisition and trafficking across the cell envelope of Gram-positive bacteria. Group A streptococcus (GAS), a β-haemolytic human pathogen, expresses a NEAT protein, Shr, which binds several haemoproteins and extracellular matrix (ECM) components. Shr is a complex, membrane-anchored protein, with a unique N-terminal domain (NTD) and two NEAT domains separated by a central leucine-rich repeat region. In this study we have carried out an analysis of the functional domains in Shr. We show that Shr obtains haem in solution and furthermore reduces the haem iron; this is the first report of haem reduction by a NEAT protein. More specifically, we demonstrate that both of the constituent NEAT domains of Shr are responsible for binding haem, although they are missing a critical tyrosine residue found in the ligand-binding pocket of other haem-binding NEAT domains. Further investigations show that a previously undescribed region within the Shr NTD interacts with methaemoglobin. Shr NEAT domains, however, do not contribute significantly to the binding of methaemoglobin but mediate binding to the ECM components fibronectin and laminin. A protein fragment containing the NTD plus the first NEAT domain was found to be sufficient to sequester haem directly from methaemoglobin. Correlating these in vitro findings to in vivo biological function, mutants analysis establishes the role of Shr in GAS growth with methaemoglobin as a sole source of iron, and indicates that at least one NEAT domain is necessary for the utilization of methaemoglobin. We suggest that Shr is the prototype of a new group of NEAT composite proteins involved in haem uptake found in pyogenic streptococci and Clostridium novyi.
Acquisition of iron from host sources is of vital importance to many pathogenic bacteria during the course of infection. Mammalian hosts limit the availability of free extracellular iron to levels around 10−18 M (Bullen, 1981) by producing iron-chelating proteins such as lactoferrin and transferrin or by storing it in ferritin within the cells. However, nearly 75% of the iron in the human body is found in the form of haem, where it is incorporated into the protoporphyrin ring and serves as a prosthetic group of haemoglobin, myoglobin and some enzymes (Stojiljkovic and Perkins-Balding, 2002; Tong and Guo, 2009). The process of obtaining haem from the host by Gram-positive pathogens often involves binding of haem or haemoproteins by bacterial receptor proteins which then deliver the haem to a membrane-bound ABC transporter for translocation to the cytoplasm (Wandersman and Delepelaire, 2004; Tong and Guo, 2009). The first Gram-positive haem transporter to be described was the hmu (haemin/haemoglobin utilization) system of Corynebacterium diptheriae (Drazek et al., 2000). The hmuTUV genes share homology with Gram-negative haem-ABC transporters such as found in Yersinia pestis (Hornung et al., 1996; Thompson et al., 1999). HmuT, which is localized to the cell membrane, binds haemin or haemoglobin directly; HmuU and V are the membrane permease and the ATPase respectively. Two membrane-anchored haem receptors, HtaA and HtaB, encoded by the hmu chromosomal locus were recently described and hypothesized to work in conjunction with the HmuTUV transporter in haem acquisition and transport across the cell envelope (Allen and Schmitt, 2009). Inside the Corynebacterium cytoplasm, a haem-oxygenase enzyme, HmuO, degrades the haem and releases the iron for use by the pathogen (Wilks and Schmitt, 1998; Kunkle and Schmitt, 2007).
The haem uptake system studied in the most detail in Gram-positive bacteria is that from Staphylococcus aureus and is termed the Isd (iron-regulated surface determinants) system. In the Isd system, the IsdA, B and H (HarA) proteins are covalently attached by the SrtA sortase enzyme to the cell wall, where they interact with a variety of ligands including haem, haemoglobin, haemoglobin–haptoglobin complex, fibrinogen and fibronectin (Dryla et al., 2003; Mazmanian et al., 2003; Clarke et al., 2004; Torres et al., 2006). These proteins obtain haem and deliver it across the cell wall and the cell membrane in a cascade fashion via IsdC and the IsdEF ABC transporter (Skaar et al., 2004; Skaar and Schneewind, 2004; Wu et al., 2005). Unlike the surface-exposed Isd receptors, IsdC is embedded deep in the cell wall by a dedicated sortase, SrtB (Mazmanian et al., 2002; 2003; Marraffini and Schneewind, 2005). Ligand binding by IsdA, B, C and H (HarA) is mediated by NEAT (Near Transporter) domains, which are found in one or more copies in each of the receptor proteins.
Haem uptake has also been studied in group A streptococcus (GAS), a β-haemolytic pathogen that uses host haem-containing proteins as an iron source (Eichenbaum et al., 1996). One system involves a 10-gene iron-regulated operon that has been termed Sia (streptococcal iron acquisition) (Bates et al., 2003). Genes three through five in the cluster [siaABC or htsABC (Lei et al., 2003)] encode an ABC transporter that shares significant homology with haem or siderophore transporters found in other bacterial species (Bates et al., 2003). The second gene in the operon, shp, encodes a surface protein with β-sandwich fold similar to that of NEAT domains and is considered a distal member of the NEAT family (Aranda et al., 2007). Shp protein has been reported to bind haem at the cell surface and transfer it to SiaA (HtsA), the lipoprotein component of the ABC transporter (Lei et al., 2003). The haem co-ordination of SiaA has recently been elucidated by further biophysical studies and is described as six-co-ordinate and low-spin, employing methionine and histidine as axial ligands (Sook et al., 2008). The first gene in the sia operon encodes the Shr (streptococcal haemoprotein receptor) protein. Shr is a large (145 kDa) hydrophilic protein that does not share significant overall homology with known haem or haemoprotein receptors (Bates et al., 2003). The first studies of Shr revealed that it plays a role in iron acquisition. It was observed to bind haemoglobin and haemoglobin–haptoglobin complex (Bates et al., 2003). The transfer of haem from Shr to the protein Shp has also been described (Zhu et al., 2008). Shr was recently demonstrated to bind the extracellular matrix (ECM) proteins fibronectin and laminin, suggesting that it also acts as an adhesin (Fisher et al., 2008). A null shr mutant is attenuated for virulence in a zebrafish model for necrotizing fasciitis, underscoring the importance of Shr to the infection process in vivo (Fisher et al., 2008).
Shr has two NEAT domains, but its overall domain architecture, which includes a unique N-terminal domain (NTD) and a series of leucine-rich repeats, is different from any of the characterized haem and haemoprotein-binding receptors, including the NEAT-containing Isd proteins of S. aureus. Shr also lacks a cell wall-anchoring motif typical of the Isd receptor proteins of S. aureus; at the C-terminus, Shr has a hydrophobic segment with a positively charged tail that threads the protein through to the cytoplasmic membrane. It was recently demonstrated that Shr spans the cell wall and is exposed to the extracellular environment (Fisher et al., 2008). Shr is proposed to participate in the acquisition of haem by GAS and its delivery to Shp and/or the SiaABC transporter. The uptake of haem from haemoproteins by Shr or its direct role in iron acquisition has not been shown, however. In this study we establish the function of Shr in haemoglobin use and haem uptake by GAS. We analyse the functional domains of this receptor protein and present evidence that the mechanism of haem uptake by Shr is different from that of the characterized Isd proteins. We suggest that Shr is a prototype of a new group of NEAT proteins involved in haem uptake.
To distinguish between the two NEAT domains in Shr, the closest NEAT domain to the amino group will be referred to as NEAT1 and the second NEAT domain from the amino group will be referred to as NEAT2.
Shr is a composite NEAT protein found in pyogenic streptococci and Clostridium novyi
NEAT domains are key ligand-binding domains used by receptor proteins involved in haem acquisition and translocation in Gram-positive bacteria. We recently reported that the GAS Shr protein has two NEAT domains that are separated by an LRR region (Fisher et al., 2008). Additional sequence examination also identified an EF-hand motif between the first NEAT domain of Shr and the LRR segment (residues 532 and 544) and two copies of a short domain with unknown function, DUF1533 (residues 61–123 and 203–269) in the N-terminal region of Shr (Fig. 1). In silico analysis using the web-based SMART tool (Schultz et al., 1998; Letunic et al., 2009) reveals that there are about 160 NEAT domains in 80 proteins encoded by Gram-positive bacteria from the Firmicutes phylum. Most of these NEAT proteins are Isd-like molecules, which contain one or more copies of NEAT domains, a leader peptide, and in some cases a sortase recognition signal or other type of cell wall-binding region. A few proteins consist of an LRR segment in addition to export signals and NEAT domain(s); these include the haem uptake protein of B. cereus, IlsA (Daou et al., 2009), and the following hypothetical proteins A0PYT7 (Clostridium novyi), O6HLL6 and O6HNR0 (Bacillus thuringensis) and 073BH4 (B. cereus). Shr appears to be the first characterized protein with DUF1533 domains. An examination of the database demonstrated that DUF1533 is found in duplication in putative proteins with unknown function from the Clostridia class and in two species of Paenibacillus. Therefore, the domain architecture of Shr is different and more complex than most of the previously described NEAT receptors or the hypothetical NEAT proteins found in bacterial genomes. The complex domain arrangement of Shr is intriguing and suggests that it evolved by joining several domains found separately in bacterial proteins of Firmicutes. Shr orthologues, which share identical or nearly identical domain architecture, are found in C. novyi as well as in the pyogenic streptococci Streptococcus equi zooepidemicus and S. dysgalactiae (Fig. 1). An shr orthologue is found in the genome of S. equi ssp. equi as well; however, a frameshift mutation results in a truncated protein (Holden et al., 2009). All streptococcal Shr orthologues are closely related in their primary amino acid sequence (58–86% identity) while the C. novyi orthologue shares fewer identical residues (∼30%). Intriguingly, the shr gene in all the streptococci is part of a 10-gene cluster which is homologous to the sia operon of GAS. The shr gene in C. novyi on the other hand is found in a genomic locus that encodes only a putative LRR-NEAT protein (A0PYT7). Together these observations suggest that Shr may represent a new type of composite NEAT protein family.
Shr obtains haem from solution and reduces ferric haem to ferrous haem
Shr binding to haemoproteins in vitro and its genomic location in the sia operon together with the haem-binding protein, Shp, and the SiaABC haem transporter suggest that Shr is involved in haem acquisition and transport by GAS (Bates et al., 2003). This hypothesis was recently supported by the observation that purified Shr transfers haem to Shp (Zhu et al., 2008). The sequestering of haem from host proteins by Shr was not previously demonstrated, however, and the mechanism of haem and haemoprotein binding has not been investigated. To characterize haem uptake by Shr, a histidine-tagged recombinant protein (rShr) (Bates et al., 2003) was prepared and analysed (Fig. 2A). Shr is readily reduced following treatment with dithionite or oxidized by ferricyanide, producing the corresponding absorption spectra with maxima around 410 nm for the bound ferric haem or 430, 540 and 560 nm for the ferrous haem (Zhu et al., 2008). In this study we found that the spectral properties of rShr after ferricyanide treatment were almost identical to those without treatment, with Soret bands at 412 and 414 nm (Fig. 2B green and blue lines respectively). On the other hand, the addition of d,l-dithiothreitol (DTT) resulted in a shift in the absorption peak to 427 nm (Fig. 2B red line) and the production of more resolved peaks at 536 and 566 nm, spectral characteristics of ferrous haem–protein complexes (Makinen, 1983). Therefore, our data indicate that rShr was purified from Escherichia coli as mostly ferric haem complex. In the course of Shr purification, we observed that haem-bound rShr was considerably more stable than the haem-free protein, and that the addition of haemin to the E. coli culture prior to harvesting the cells resulted in a higher production of intact rShr (data not shown).
Haem binding by rShr was investigated further by monitoring the changes in the UV-visible spectrum following the addition of increasing amounts of haemin (haem with ferric iron) to the protein solution. The addition of free haemin resulted in a concomitant increase in rShr-bound haemin, as was indicated by the growing absorbance at 414 nm (Fig. 2C). Surprisingly, the UV-visible spectrum of rShr following the haemin addition also revealed growing absorption peaks at 427, and at 540 and 564 nm as well (Fig. 2C insert). Removing the free haem by dialysis did not lead to changes in the spectrum (data not shown). The growing peaks at 427, 540 and 564 nm indicate a simultaneous increase in rShr-bound ferrous haem. The addition of haemin to rShr was done under aerobic conditions (using an open tube and vigorous mixing) and in the absence of reducing agents. Therefore, the observed rise in rShr-bound haem suggests that Shr reduces the added haemin. The addition of ferricyanide to the protein solution following titration with 20 µM haemin resulted in a shift of the absorption peak from 427 back to 410 nm. The 410/280 absorbance ratio that is indicative of the ferric-haem load in Shr (Zhu et al., 2008) was changed from 0.59 to 0.75. Oxidation with ferricyanide also eliminated the peaks at 540 and 564 nm (Fig. 2D). Therefore, ferricyanide was able to oxidize the ferrous iron of the Shr-bound haem, confirming that the changes in Shr spectrum seen following the addition of free haemin were due to a reversible reduction of the protein-bound haemin. It was previously reported that Shr could be purified from E. coli as a mixture of ferric and ferrous iron haem (Zhu et al., 2008). Our observations suggest that Shr has an inherent ability to reduce the ferric haem and to provide a stable environment for the produced ferrous complex. The autoreduction activity is a very intriguing characteristic of Shr. To the best of our knowledge, it is the first report of haem reduction by a bacterial haem receptor.
NEAT1 and NEAT2 are both haem-binding domains in Shr
Haem binding by the Isd proteins is imputable to their NEAT domains (Grigg et al., 2010). However some NEAT domains have been reported to not bind haem. For example, IsdH NEAT3 domain binds haem (Tiedemann et al., 2008; Watanabe et al., 2008; Pilpa et al., 2009), whereas IsdH NEAT1 and IsdH NEAT2 domains interact only with haemoglobin and haptoglobin (Pilpa et al., 2009). Isd NEAT domains hold the haem within a hydrophobic pocket through several conserved residues including two invariant tyrosines, one of which co-ordinates the iron (Tyr-166 in IsdA) and a second residue (Tyr-170 in IsdA) that interacts with both the haem pyrrole ring and the co-ordinating tyrosine (Grigg et al., 2007). Sequence analysis revealed that not all of the conserved residues in the Isd haem binding sites are found in Shr NEAT domains (Fig. S1); most significantly, both of the NEAT domains in Shr are missing the iron-co-ordinating residue Tyr-166, and only NEAT1 has the Tyr-170. Interestingly, the second haem-binding protein coded by the sia operon, Shp, does not use tyrosine residues to co-ordinate the haem iron, and instead utilizes two methionines (Aranda et al., 2007).
To investigate haem acquisition by Shr, several recombinant proteins containing one or more of the component domains of Shr were constructed with an N-terminal fusion to the Strep-tag epitope. These Shr variants include recombinant proteins with the N-terminal domain of Shr (NTD) or the N-terminal domain through NEAT1 (NTD-N1), the NEAT1 domain, and the NEAT2 region (Fig. 3A). The recombinant proteins were overexpressed, purified by FPLC using a Strep-Tactin column and analysed. The protein containing only the NEAT1 region turned out to be highly insoluble and was therefore excluded from further investigations. SDS-PAGE analysis confirmed the production and purification of the recombinant Shr protein fragments, revealing protein bands at the expected size for each construct: 61 kD (NTD-N1), 42 kD (NTD) and 23 kD (NEAT2, Fig. 3B).
The Shr protein variants that contained the first NEAT domain (NTD-N1) or the second NEAT domain (NEAT2) both had red colour when purified and a UV-visible spectrum consistent with bound haem. The purified NTD-N1 protein showed a significant absorbance at 410 nm indicating that it was co purified with ferric haem (Fig. 3C). In contrast, the optical spectrum of the purified NTD fragment showed no band at the Soret region indicating that it did not contain haem (Fig. 3D). To test the haem binding ability by Shr's N-terminal region, the recombinant NTD protein was incubated with increasing concentrations of haemin. The optical spectrum of the protein after incubation showed no absorption at the Soret region. Instead, similar to the spectrum of free haem (Fig. S2), a broad peak at 390 nm was formed as increasing amounts of haemin were added, indicating an accumulation of unbound haem in the NTD protein solution (Fig. 3E). Therefore, the NEAT1 domain contained within the NTD-N1 protein is responsible for the ferric haem binding observed by this protein fragment.
Unlike the ferric-haem load of the NTD-N1 protein, the optical spectrum of the purified NEAT2 protein suggests that it is purified with a mixture of ferric and ferrous haem. NEAT2 spectra consistently had a significant Soret band at 428 nm along with sharp peaks at 535 nm and 564 nm. However, variations in the intensity of absorbance at 410 and 428 nm (indicating different ratio of the protein bound ferrous and the ferric forms) were observed in the spectrum of NEAT2 from different protein preps (Fig. 3F and the blue line in Fig. S3). To further investigate haem binding by NEAT2, the protein was titrated with increasing concentrations of free haemin and the UV-visible spectrum was monitored (Fig. S3). As observed with the full-length Shr, the addition of free haemin resulted in concomitant increase of absorption at around 410 nm as well as 428, 535 and 564 nm. NEAT2 was then treated with ferricyanide and the UV-visible spectrum was taken after 5 and 30 min intervals and after 24 h. Within 5 min after addition of ferricyanide (Fig. 3G red line), the absorbance at ∼410 nm increased compared with NEAT2 without ferricyanide (Fig. 3G blue line), and the bands at ∼428, 535, 564 nm were almost gone, indicating that the NEAT2 protein was mostly in the oxidized form. The spectra did not change significantly at 30 min (data not shown). After 24 h (Fig. 3G green line), a red shoulder on the Soret band was seen, and the absorbance at 428, 535 and 564 nm had increased. Together these observations indicated that the NEAT2 protein autoreduced slowly.
Methaemoglobin binding is mediated by the NTD of Shr, which specifically recognizes the holo form
Following erythrocyte lysis, the α2β2 heterodimeric haemoglobin converts to methaemoglobin, in which the haem is found in the ferric form. This is largely an αβ heterodimer (Ascenzi et al., 2005; Umbreit, 2007). Methaemoglobin is likely to be a physiologically relevant haem source for the haemolytic GAS. To investigate Shr interactions with methaemoglobin, we developed and used an enzyme-linked immunoabsorbent assay (ELISA). rShr and the recombinant Shr fragments NTD-N1, NEAT2 and NTD were used to coat the wells of microtitre plates and allowed to interact with increasing concentrations of methaemoglobin (Fig. 4A). Ligand binding by the immobilized proteins was detected using haemoglobin antiserum. Wells coated with BSA and uncoated wells were used as controls for non-specific interactions. rShr bound methaemoglobin in a dose-dependent and saturable manner, while only low background binding (OD405≤ 0.1) of the haemoglobin antiserum to the control wells was observed. The recombinant Shr fragments NTD and NTD-N1 bound methaemoglobin with binding profiles that were similar to the full-length Shr, and methaemoglobin binding appeared saturated at a concentration of 10 nM (Fig. 4A). The observation that the full-length Shr, NTD-N1 and NTD alone equivalently bind methaemoglobin indicates a preponderant role for the NTD in haemoglobin binding. In contrast, no methaemoglobin binding by the NEAT2 protein was detected. Therefore, unlike IsdA, IsdB and IsdH proteins, which use NEAT domains to bind haemoglobin, an uncharacterized protein pattern found in the NTD of Shr interacts with haemoglobin.
We hypothesize that Shr interacts with methaemoglobin to acquire haem from the host. We therefore asked whether Shr could differentiate between the apo and the holo forms of haemoglobin. Haem was removed from methaemoglobin according to Asakura et al. (1964) and the formation of the apoprotein was confirmed by the UV-visible spectrum, which revealed no absorption at the Soret region (data not shown). The binding of NTD to immobilized apohaemoglobin was tested by ELISA. In contrast to its interactions with holoprotein, no binding of NTD to apohaemoglobin was observed (Fig. 4A, red line). Similar to the NTD, the full-length Shr did not bind apohaemoglobin (Fig. S4). A control ELISA performed with immobilized holo and apohaemoglobin demonstrated that the haemoglobin-specific antibody was able to detect both forms of haemoglobin similarly over the range of haemoglobin concentrations studied (Fig. 4B). Therefore, the absence of binding of apohaemoglobin in the experimental ELISA shown in Fig. 4A (red line) was not due to a lack of ability of the haemoglobin antiserum to recognize the apoprotein. In conclusion, these experiments demonstrate that Shr differentiates between the holo and the apo forms of haemoglobin and binds only to haem-loaded protein.
The NEAT2 domain in Shr mediates most of its binding to ECM
We have recently observed that in addition to its interactions with haemoproteins, Shr also functions as an adhesin and binds fibronectin and laminin (Fisher et al., 2008). To determine the domains involved in the ability of Shr to bind these proteins components of the ECM, immobilized rShr, NTD, NTD-N1 and NEAT2 were allowed to react with the ECM components using ELISA. Ligand binding was detected with antibodies specific for fibronectin or laminin. When fibronectin was added in increasing concentrations to the immobilized proteins, rShr as well as the recombinant fragments NTD-N1 and NEAT2 bound it in a concentration-dependent and saturable manner (Fig. 5A). The NEAT2 protein demonstrated the highest binding to fibronectin, while only low level binding was seen with NTD-N1. No interactions with fibronectin were demonstrated by the immobilized NTD protein. These observations suggest that NEAT regions mediate the observed Shr binding to fibronectin. Similar observations were made with laminin; as shown on Fig. 5B, rShr, and NEAT2 proteins bound laminin, while no significant binding to laminin was observed by the NTD or NTD-N1. Together, these observations indicate that while both Shr NEAT domains are able to interact with some ECM components, the NEAT2 domain plays a more significant role in this activity of Shr.
The NTD-NEAT1 region of Shr is sufficient for haem acquisition from methaemoglobin
We next asked if the NTD-N1 fragment of Shr, which contains the haemoglobin-binding region and the haem-binding NEAT1, is sufficient for haem acquisition from methaemoglobin. Haem was removed from the purified NTD-N1 (Asakura et al., 1964), and the formation of apo NTD-N1 was confirmed by UV-visible spectrum analysis (Fig. 6A). The haem transfer assay was performed over a Strep-Tactin column with immobilized apoNTD-N1 protein. Methaemoglobin in equimolar amounts to the immobilized apoNTD-N1 protein was flowed through the column. The bound haemoglobin was removed by extensive washes with salt containing buffer (see Experimental procedures), and the NTD-N1 protein was then eluted with desthiobiotin. Western blot analysis of the fractions collected during this procedure revealed that the haemoglobin containing fractions also included low amounts of NTD-N1 in addition to methaemoglobin (lane 3, Fig. 6B), suggesting that some methaemoglobin/NTD-N1 complexes were washed from the column. The NTD-N1 fraction that was eluted with desthiobiotin, however, did not contain a detectable amount of haemoglobin (lane 4, Fig. 6B). The optical absorbance of NTD-N1 after the methaemoglobin passage showed a sharp peak at 411 nm, indicating that the apoNTD-N1 acquired haem from methaemoglobin (red line, Fig. 6A). To confirm that the observed absorbance at 411 nm resulted from NTD-N1/haem complex and not from trace amounts of methaemoglobin, we analysed the absorbance of 0.7 µM methaemoglobin solution, a concentration that is above the detection level of the haemoglobin antibody utilized in the assay (lane 6, Fig. 6B). This analysis revealed that methaemoglobin, at the tested concentration, had a significantly lower Soret band in than the NTD-N1 fraction (data not shown).
A similar experiment was performed with immobilized NTD-N1 with haemin chloride solution (in fourfold molar excess) instead of methaemoglobin in the mobile phase. The optical analysis of NTD-NEAT1 after passage of free haemin also revealed that it acquired haem as indicated by the peak at 412 nm (blue line Fig. 6A). However the peak at the Soret region of NTD-N1 after the passage of methaemoglobin was significantly higher than that after contact with the haemin chloride solution. This observation suggests that the NTD-N1 protein acquires more haem from haemoglobin than from the haemin solution, supporting haem transfer from methaemoglobin directly to the immobilized NTD-N1. Haem acquisition by apoNTD-N1 was also investigated using an alternative assay in which the NTD-N1 was allowed to interact with methaemoglobin in solution at room temperature. The UV-visible spectrum of the NTD-N1 protein after its separation from methaemoglobin (using Strep-Tactin column) revealed a sharp peak at 410 nm (Fig. S5), demonstrating haem transport from methaemoglobin to the NTD-N1 protein. Interestingly, the absorption intensity at the Soret region following 5 min of co-incubation was about 80% of that seen following 75 min, indicating rapid haem sequestering by the NTD-N1 protein.
Shr is required for GAS growth using haemoglobin as the sole iron source
To determine the importance of the haem binding domains to the function of Shr in vivo, several GAS mutants containing in-frame deletions of various regions in shr were constructed. These include a mutant with NEAT1 deletion mutant (ΔNEAT1), a mutant with a deletion that spans the distal part of the LRR and most of the second NEAT domain (ΔNEAT2), and a mutant with a large deletion that includes both NEAT domains and the region in between (ΔNEAT1-2, Fig. 7A). The production of shr alleles in the expected size in the genome of each of the mutants and of the corresponding Shr proteins was confirmed by PCR and Western blot analyses (Fig. 7B and C). This analysis also confirmed the production of the wild-type (wt) Shr when the ΔNEAT1-2 was complemented. Successful complementation of the null shr mutant was previously shown (Fisher et al., 2008). RT-PCR analysis with primers specific for siaA, which is located downstream of shr, verified that the shr mutations were not polar and did not affect the expression of the downstream genes in the sia operon (Fig. 7B).
The ability of wt, an shr null mutant (Fisher et al., 2008) and the isogenic mutant strains described above to use haemoglobin as a sole source of iron was investigated using a growth assay that is based on iron-depleted chemically defined medium (CDM). The wt strain and all of the shr mutants grew well in complete CDM medium containing 20 µM of free iron (Fig. 7D). On the other hand, CDM that was prepared without iron and contained 2 mM of the ferric chelator 2,2-dipyridyl did not support significant growth of any of the tested GAS strains (Fig. 7E). The addition of 20 µM methaemoglobin to the iron-depleted CDM restored growth of the wt strain to the level obtained with CDM containing 20 µM iron (Fig. 7F); lower haemoglobin concentration, however, did not support growth of any of the strains in the iron-depleted medium (Fig. S6A and B). These observations demonstrate that iron is indeed the limiting factor for GAS growth in the 2,2-dipyridyl CDM and that GAS is able to use haemoglobin to satisfy its iron needs as we previously reported (Eichenbaum et al., 1996). No significant growth differences were observed between the shr mutants containing a deletion of NEAT1 (ΔNEAT1) or of NEAT2 (ΔNEAT2) and the wt strain in the 2,2-dipyridyl CDM supplemented with methaemoglobin. Therefore, the ΔNEAT1 and the ΔNEAT2 shr mutants are not affected in their ability to use haemoglobin as a source of iron. On the other hand, the growth of the shr null mutant and to a lesser extent that of the mutant that was missing both of the NEAT domains (ΔNEAT1–2) was impaired (Fig. 7F). The growth phenotype demonstrated by the shr mutants was reversed by complementation with the shr gene (Fig. 7G). These findings establish that Shr is required for haemoglobin utilization in vivo and suggest that Shr function requires at least one of the haem-binding NEAT domains. The addition of methaemoglobin in higher concentration (60 µM) supported better growth of the tested GAS strains (Fig. S6C). Thus, the Shr-dependent pathway for haemoglobin utilization in GAS may be of high affinity. However, additional pathways for acquisition of iron from haemoglobin also exist, as previous findings suggest (Montañez et al., 2005).
During the infection process, the β-haemolytic GAS can tap into the intracellular haem reservoir due to the potent haemolysins it produces and satisfy its needs for iron with haemoglobin, haemoglobin–haptoglobin and other haem-containing proteins (Francis et al., 1985; Eichenbaum et al., 1996). Previous observations implicated the surface-exposed NEAT protein, Shr, in the first step of haem acquisition from host proteins by GAS (Bates et al., 2003; Zhu et al., 2008). In this work we have provided the first direct support for this proposition by demonstrating that Shr can obtain haem from methaemoglobin and by establishing that Shr function is important for GAS ability to use methaemoglobin as an iron source. GAS use of Shr in haem acquisition is reminiscent of NEAT-containing receptors such as the Isd proteins in S. aureus and related proteins from other Gram-positive bacteria. In this study, however, we demonstrate that GAS Shr structure and function are different from that of previously characterized NEAT proteins and suggest that Shr represents a new type of protein family with a different mode of haemoglobin binding and haem acquisition.
Shr represents a family of composite NEAT proteins
Shr is a complex NEAT protein that consists of a unique combination of domains and protein motifs (Fig. 1). The database contains many secreted or surface proteins with NEAT domain(s); a few also carry LRR region(s) including IlsA of B. cereus (Daou et al., 2009). LRR are commonly involved in protein recognition and protein–protein interactions (Kobe and Kajava, 2001). It is possible that the LRR may help facilitate the intra-molecular communications that are likely to take place in Shr or its interactions with the other transport components such as Shp. DUF1533 is a domain of unknown function found in hypothetical proteins that contain secretion or export signals and sometimes other functional regions including LRR. The combination of two DUF1533, a LRR region and two NEAT domains is seen for the first time in Shr, however. Interestingly, Shr seems to be the first characterized NEAT protein that has an EF-hand motif (located between the NEAT1 and the LRR regions, Fig. 1). The EF-hand motif is a calcium-binding domain that is ubiquitous among eukaryotic calcium-binding proteins such as calmodulin, but is also found in bacterial sequences (Michiels et al., 2002; Rigden et al., 2003; Zhou et al., 2006). As with other bacterial proteins with EF-hands, the functional significance of this motif is yet to be determined. Further in silico analysis identified several orthologues of Shr in C. novyi, S. equi ssp. zooepidemicus, S. equi ssp. equi and S. dysgalactiae. These Shr-like proteins share with Shr significant sequence homology and domain architecture, suggesting that GAS Shr is a representative of a small family of NEAT proteins that evolved by combining domains found in surface or secreted proteins encoded by bacteria from phylum Firmicutes.
Shr is the only NEAT protein to show ferric haem reduction
We isolated rShr with ferric haem (Fig. 2B), indicating that this protein can sequester haem from E. coli. It was previously reported that Shr could be isolated from E. coli apoprotein with a mixture of ferric and ferrous haem (Zhu et al., 2008). When we titrated the protein with free haemin, the increase in the absorption around 414 nm demonstrated that Shr can also acquire ferric haem from solution (Fig. 2C). The concurrent rise in absorption at 427, 540 and 564 nm indicated increasing amounts of rShr-bound ferrous haem and was reversed by oxidation (by ferricyanide, Fig. 2D). This increase in bound ferrous haem following the addition of ferric haem to the purified rShr strongly suggests that Shr autoreduces the haem, even in air. Shr clearly provides a stable environment for the bound ferrous haem. This is the first demonstration that Shr can obtain haem from solution and reduce it to ferrous haem. Haem reduction is a unique property of Shr that to our knowledge has not been reported for any other NEAT protein.
The iron oxidation state in protein-bound haem can have a significant impact on the haem fate and its environment. For example, reduction of the ferric haem in IsdC NEAT domain results in the loss of the haem from the protein (Pluym et al., 2008). On the other hand, the full-length IsdA and its single NEAT domain can bind both ferric and ferrous haem. The reduction to ferrous haem, however, changes the iron axial ligand from tyrosine to histidine and renders it accessible to small anionic ligands such as CO. Based on these observations, it is suggested that iron oxidation/reduction lead to subsequent conformational changes with closing/opening of the haem-binding pocket in IsdA (Vermeiren et al., 2006; Pluym et al., 2008). Our in vitro study of rShr shows that the full-length protein is able to bind the haem group in both the ferric and the ferrous forms as well. It seems possible that, as in IsdA however, iron oxidation/reduction may be accompanied by structural changes that influence the haem location within the protein (i.e. binding to first or the second NEAT domain) or the subsequent in vivo steps in the haem trafficking such as the haem transfer to Shp or SiaA.
Functional and sequence analysis show that both NEAT domains in Shr are divergent from one another and from the NEAT domains of the Isd protein family
The optical spectrum of the Shr variants NTD-N1 and NEAT2 demonstrate that they both complex haem, while the NTD protein, in contrast, did not show any haem binding. Sequence alignment demonstrated that only a few of the residues that contact the haem in Isd NEAT domains are conserved in the putative haem biding sites of NEAT1 and NEAT2 in Shr (Grigg et al., 2010) (Fig. S1). Most noticeably, both of the NEAT domains in Shr are missing the potential iron-co-ordinating residue, Tyr-166, a haem ligand in other NEAT proteins, and only NEAT1 has Tyr-170 (proposed to regulate haem binding and release) (Fig. S1). Therefore, the haem-binding region in both Shr NEAT domains is quite different from that of Isd-like haem-binding NEAT domains and the haem iron may not be co-ordinated by a tyrosine, at least in the NEAT2 domain of Shr. These observations suggest that Shr NEAT domains have different mechanism for interactions with haem. Likewise, Shp, which can acquire haem from Shr, has a unique iron co-ordination involving two methionines within the single Shp molecule that function as axial ligands (Aranda et al., 2007). Additional work is needed to determine the haem axial ligands of the NEAT domains in Shr. Future investigations of the evolutionary relation between Shr NEAT domains and Shp, which represents a more remote member of the NEAT family, are warranted.
Both of the NEAT domains in Shr bind haem. The optical spectra of NTD-N1 and NEAT2 proteins as isolated from E. coli indicate that NEAT1 is complexed with ferric haem, and that NEAT2 is bound to both ferric and ferrous haem (Fig. 3C and F). Like with the full-length Shr, titration of NEAT2 with increasing concentration of haemin resulted in raising amounts of bound ferric and ferrous haem (Fig. S3). Over time some of the ferric haem in NEAT2 (produced by oxidation with ferricyanide) was reduced to ferrous haem (Fig. 3G). Together these observations suggest that the NEAT2 domain in Shr is capable of autoreduction. Functional differences between NEAT1 and NEAT2 were also found in their interaction with non-haem ligands. While Shr NEAT2 interacts with both fibronectin and laminin molecules, NEAT1 binding to fibronectin is significantly weaker and it does not demonstrate significant binding to laminin (Fig. 5).
A unique site in Shr N-terminal region mediates binding to methaemoglobin
We demonstrated in this study, using ELISA with immobilized Shr variants, that the full-length rShr, the NTD and the NTD-N1 all bound methaemoglobin similarly. The NEAT2 protein, on the other hand, did not interact with methaemoglobin (Fig. 4A). Therefore, it appears that an unspecified region within Shr NTD mediates its binding to methaemoglobin. While this manuscript was in preparation Meehan et al. (2010) reported that like GAS NTD, the truncated Shr molecule produced by S. equi ssp. equi (consisting mostly of Shr NTD) binds haemoglobin and haemoglobin–haptoglobin complex. The finding that the interaction of Shr with methaemoglobin is not mediated by NEAT domains distinguishes Shr from the previously studied Isd receptors. Domain analysis of IsdA and IsdH demonstrated that binding to host haemoproteins is carried out by the single NEAT domain in IsdA and two of the NEAT domains of IsdH (Clarke et al., 2004; Pilpa et al., 2009). Site-directed mutagenesis localized the binding to a conserved aromatic motif also found in the first NEAT domain of the IsdB (Pilpa et al., 2009), which binds haemoglobin and haemoglobin/heptoglobin complex (Torres et al., 2006). Consistent with the results of the ELISA experiments, both of the Shr NEAT domains are missing the haemoglobin binding sites identified in the Isd proteins. Additional work is required to identify the region within Shr NTD that is involved in haemoglobin binding. The sequence of the Shr N-terminal region is unique, and besides the two copies of DUF1533, it shares sequence homology only with Shr orthologues. Therefore, haemoglobin binding is mediated by a new protein motif in Shr.
Haemoglobin binding by bacterial receptors is not fully understood. In this work we show that the Shr NTD binds only haem-containing haemoglobin (Fig. 4A). The functional significance of this observation is that Shr may release the bound haemoglobin after sequestering all the haem. It is not clear how Shr differentiates between the apo and the holo forms of methaemoglobin. Recognition of the haem moiety does not seem to be part of Shr binding to methaemoglobin as NTD does not bind haem and the haem is mostly buried within haemoglobin (Genco and Dixon, 2001). We hypothesize therefore that Shr recognizes a tertiary structure in the holoprotein that is disrupted when haem is lost, rather than recognizing a linear region within the α or β polypeptides of haemoglobin.
The NTD and NEAT1 in Shr are sufficient for haem acquisition from methaemoglobin in vitro
It was previously demonstrated that purified Shr transfers haem directly to apoShp in vitro (Zhu et al., 2008), while the direct movement of haem from methaemoglobin to Shp was not observed. Therefore it was hypothesized that Shr is involved in the first step of haem sequestering from haemoglobin. In this study we used the column-immobilized apoNTD-N1 to ask if it could obtain haem following transient interactions with methaemoglobin or free haem (Fig. 6). Spectral analysis performed with NTD-N1 following the passage of methaemoglobin showed that it obtained haem. In a separate assay, co-incubation of apoNTD-N1 with methaemoglobin in solution demonstrated that the haem transfer from methaemoglobin is fast. NTD-N1 was also able to receive haem from solution, but to a lesser extent. These experiments imply that haem is transferred directly from methaemoglobin to the NTD-N1 protein. Further investigations are required to determine if NEAT2 can obtain haem from NEAT1 and/or from methaemoglobin, and to find out which of the Shr domains are required for the subsequent step in haem trafficking.
Shr is needed for haem uptake from methaemoglobin in vivo
The growth of the shr- and ΔNEAT1–2 mutants in iron-depleted medium supplemented with haemoglobin was impaired in comparison to that of the wt GAS strain (Fig. 7F). The mutant growth phenotypes were reversed by complementation with the shr gene, establishing the role of Shr in haemoglobin utilization in vivo. The residual growth of the ΔNEAT1–2 in low haemoglobin concentration and the full growth of both mutants observed when higher amounts of haemoglobin were added (Fig. S6C) are consistent with a previous report suggesting that additional haemoglobin utilization pathways are found in GAS (Montañez et al., 2005). Since the shr mutants required a higher concentration of haemoglobin to restore growth than the wt strain, we suggest that Shr mediates a high-affinity pathway. It is noteworthy, however, that the deletion of a single NEAT domain (as in the ΔNEAT1 and ΔNEAT2 mutants) did not have significant growth defect. These findings indicate that either one of the NEAT domains is sufficient for haem uptake from haemoglobin in vivo, suggesting that despite the differences found between Shr NEAT domains, they have some functional redundancy.
In conclusion, this study establishes the role of Shr in haem acquisition from haemoglobin, and demonstrates that the streptococcal receptor is a representative of a structurally and functionally distinct NEAT protein family found in C. novyi and pyogenic streptococci. We have begun to elucidate the functional domains of Shr, although additional investigations are required to fully understand the mechanism of haem uptake mediated by this intriguing protein.
Strains, media and growth conditions
Escherichia coli DH5α and XL1 blue were used for cloning and gene expression. The GAS strain used in this study was NZ131, an M type 49; ZE4912, an isogenic strain with a non-polar, null mutation in shr (shr::aad9) (Fisher et al., 2008); ZE4924, a merodiploid strain, which contains both the shr::aad9 and the wt alleles of shr in the chromosome (Fisher et al., 2008). E. coli cells were grown aerobically in Luria–Bertani (LB) medium at 37°C. GAS cells were grown statically at 37°C in Todd–Hewitt broth with 0.2% w/v yeast extract (THY, Difco Laboratories) or CDM (SAFC Biosciences) as described in Montañez et al. (2005). When necessary, 100 µg ml−1 ampicillin, 100 µg ml−1 spectinomycin, 70 or 300 µg ml−1 kanamycin (for E. coli and GAS respectively) were added to the medium.
Chromosomal and plasmid DNA extraction and DNA manipulations, including restriction digest, cloning, and DNA transformation into E. coli or GAS, were performed according to the manufacturer's recommendations and with standard protocols as previously described (Sambrook et al., 1989; Eichenbaum et al., 1998). PCR for cloning was performed using the High-Fidelity AccuTaq LA DNA Polymerase (Sigma). PCR products were purified with the QIAquick PCR Purification Kit (Qiagen). DNA ligation was performed with using Fastlink ligation kit (Epicentre). For RNA extraction and analysis, GAS cells were harvested at the logarithmic growth phase and total RNA was prepared using the RiboPure-Bacteria Kit (Ambion). RNA was quantified spectrophotometrically, and its integrity was examined by agarose gel electrophoresis. For RT-PCR cDNA was produced by Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's specification. The oligonucleotide primers used in this study are listed in Table S1. Table S2 lists and describes the construction of the plasmids used in this work.
The following isogenic mutant series was constructed in NZ131 background: ZE4925 (in-frame deletion of NEAT1 in shr, ΔNEAT1), ZE4926 (in-frame deletion of the LRR 3′ and most of the NEAT2 in shr, ΔNEAT2), and ZE4929 (in-frame deletion of the region between NEAT1 up to and including NEAT2 in shr, ΔNEAT1-2). Alleles with unmarked and in-frame deletions in the shr gene with about 1 kb of flanking sequence were cloned into the temperature sensitive shuttle vector pJRS700, as described in Table S2. The mutations were then introduced into GAS chromosome by transforming NZ131 cells with each of the recombinant vectors and selecting for kanamycin resistance at 30°C. The transformants were then passed in antibiotic-free medium at 37°C. Mutants in shr gene, generated by allelic replacement via double homologous recombination were identified by screening for plasmid loss (kanamycin sensitivity). The formation of each mutation was confirmed by PCR and Western blot analysis (Fig. S5). The GAS strains ZE4925, ZE4926 and ZE4929 were engineered using plasmids pXL2, pXL13 and pXL3 respectively. The strain ZE4935 is a merodiploid containing both the wt and the ΔNEAT1-2 shr alleles in the chromosome. For ZE4935 construction, the temperature-sensitive plasmid pXL14 was introduced into ZE4929 cells and vector integration into the chromosome (via homologous recombination) was selected on kanamycin at 37°C; strain construction was confirmed by PCR and Western blot analysis (Fig. S5 and data not shown).
Overexpression and purification of recombinant Shr, NTD, and NTD-N1 and NEAT2
The expression of Strep-tag Shr (pEB2), Strep-tag Shr NTD (pEB10), Strep-tag NTD-N1 (pEB11) or Strep-tag NEAT2 (pHSL2) was induced with 200 ng ml−1 anhydrotetracycline, overnight at 27°C. Cells were harvested, resuspended in lysis buffer (100 mM Tris-HCl pH 8, 500 mM sucrose, 1 mM EDTA) with the addition of 0.5 mg ml−1 lysozyme, β-d glucopyranoside final concentration 0.5% and Complete, mini-EDTA-free protease inhibitor cocktail tablets (Roche) then lysed by sonication. The cells pellet was centrifuged and the cleared lysate was then applied to a Strep-Tactin Superflow column (IBA) with a 5 ml bed volume and purified using FPLC. A step gradient programme was used and Strep-tag proteins were eluted with five column volumes of 100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin. A cation exchange column (4 ml bed volume Hi-trap SP HP, GE Healthcare) was used for further purification of Strep-tag Shr by FPLC. The Strep-tag Shr directly after elution from the Strep-Tactin Superflow column was diluted 1:5 in 50 mM acetic acid pH 4.8, applied to the Hi-trap SP HP column and eluted with 50 mM acetic acid pH 4.8 plus 1 M NaCl2.
His-tagged Shr was expressed (pCB1) and purified as described previously (Fisher et al., 2008) with the following exceptions: when necessary, haemin in dimethyl sulphoxide was added to give a final concentration of 1 µM haemin in the cell culture 1 h before cell disruption by sonication and sonication was increased to include 10 cycles.
All proteins were prepared in Laemmli sample buffer and separated by sodium dodecyl sulphate-10% polyacrylamide gel electrophoresis (SDS-10%). Western blot analysis was performed with polyclonal antibodies against Shr raised in rabbit as described previously (Bates et al., 2003). Total protein concentration was measured using a Lowry assay (Pierce Biotechnology). Each elution fraction was stored in 15% glycerol with 200 µl of protease inhibitor cocktail (Complete, mini-EDTA-free, Roche). Fractions used for further study underwent buffer exchange to 20 mM Tris-HCl, 15% glycerol, pH 8.0 and were stored at −20°C.
Enzyme-linked immunoabsorbent assays (ELISA)
An ELISA was used to analyse the ability of Strep-tagged Shr, NTD, NTD-N1 and NEAT2 to bind to various ligands. ELISA plate wells (Costar, Corning) were coated with a 50 µl solution containing the desired concentrations of bait proteins. Wells coated with BSA and uncoated wells were used as controls for non-specific interactions. The bait proteins were diluted in PBS buffer (10 mM phosphate-buffered saline, 100 nM NaCl, pH 7.4) and included rShr, NTD, NTD-N1, NEAT2 and BSA. After the bait proteins were incubated overnight at 4°C, the wells were washed with PBS-Tween (0.05%) buffer and blocked with 200 µl of 5% soy infant formula (Nestle)-PBS-Tween for 1 h at 37°C then washed again to remove blocking solution. For apohaemoglobin preparation, haem was removed from methaemoglobin according to Asakura et al. (1964). The desired concentrations of human apo/holomethaemoglobin (Sigma), human fibronectin (BD) or mouse laminin (BD) in 5% soy/PBS-Tween were then added to each well (50 µl well−1). The wells were then washed PBS-Tween/well to remove unbound protein. Fifty microlitres of a 1:15 000 dilution of polyclonal rabbit anti-haemoglobin (sigma), rabbit anti-fibronectin (abcam) or rabbit anti-laminin (abcam) antibodies in blocking buffer were subsequently added to each well and incubated at 37°C for 1 h, and the wells were then washed. Fifty microlitres of goat anti-rabbit IgG conjugated to alkaline phosphatase (Sigma) at 1:6000 dilution in blocking buffer was added to each well and incubated at 37°C for 1 h. Interactions between recombinant Shr, NTD, NTD-N1 or NEAT2 and the ligands were then detected by adding pNPP substrate and developing the chromogenic reaction (KPN). Plates were read at 405 nm on an automated ELISA reader for intervals up to 1 h after development. An assay to assess the ability of a variety of blocking buffers to diminish nonspecific binding was also performed as described above except the ELISA wells were uncoated.
rShr, NTD, NTD-N1 and N2 UV-visible spectra
The spectrophotometric analysis of samples from 250 to 700 nm was carried out using a Varian Cary 50 Bio spectrophotometer. Absorption spectra of the purified proteins were measured on the spectrophotometer in a quartz cell with an optical path length of 10 mm. All absorption spectra shown in this study are representative of multiple experiments performed with at least three biological replicas.
Haem titration assays
A stock solution of haemin chloride in DMSO was prepared. The absorbance of a 1:1000 dilution of the stock solution at 404 nm was recorded and the concentration of haemin chloride in the stock solution was calculated using Beer's law (A = εbc where haemin in DMSO ε404 = 188 000 m−1 cm−1) (Brown and Lantzke, 1969; Collier et al., 1979). Protein samples were diluted in Strep-tag elution buffer to 3 µM. Absorbance from 250–700 nm was recorded before addition of haemin chloride. Haemin chloride was added to 1 ml aliquots of 3 µM protein to a final haemin chloride concentration of 1 µM, incubated with stirring at 4°C for 1 h and the absorbance from 250–700 nm was scanned and recorded. This was repeated for haemin chloride concentrations of 3 µM, 5 µM, 10 µM and 20 µM. Strep-tag elution buffer alone was similarly incubated with 1 µM, 3 µM, 5 µM, 10 µM and 20 µM of haemin chloride. These haem-containing buffer solutions were scanned as blanks for the UV-visible spectra of the protein solution containing corresponding concentrations of haemin chloride. The total volume of DMSO added to the protein solutions or the blank solutions ranged from 0.15 µl to 3 µl. Thus, the final DMSO concentration in the sample was 1.5 × 10−4 to 3 × 10−3 v/v. Ferricyanide and DTT treatment were performed in sealed tubes with incubation at room temperature. Ferricyanide was then removed by dialysis.
Haem transfer from methaemoglobin
Haem transfer from methaemoglobin to Shr fragment NTD-N1 was performed using FPLC. ApoNTD-NEAT1 was prepared according to the method described by Asakura et al. (1964) and 100 nmoles of the protein in 2 ml of Strep-tag wash buffer was attached to a Strep-Tactin Superflow column. Equivalent moles of methaemoglobin (100 nmoles) was flowed through the immobilized NTD-N1. The bound methaemoglobin was removed by washing several times (10 column volumes) with a wash buffer (100 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM EDTA). Subsequently, the immobilized NTD-N1 was eluted with Strep-tag elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin). Methaemoglobin flow-through and NTD-N1 elution samples were collected and analysed by Western blot using anti-Shr and anti-haemoglobin antibodies. Protein concentration in the different fractions was determined by Modified Lowry assay, and Spectroscopic analysis (250–700 nm) of 10 µM apoNTD-N1 before and after passage of methaemoglobin was carried out using a Varian Cary 50 Bio spectrophotometer.
Culture in microplate
All GAS strains were grown in CDM supplemented with 3 mM l-cysteine, 15 mM sodium bicarbonate, 2.5 mM magnesium sulphate, 44 µM calcium chloride, 15 µM zinc chloride, 20 µM manganese and either 20 µM metal iron or a range of concentrations of human haemoglobin. In the latter case, 2 mM 2,2-dipyridyl was added to the supplemented CDM to completely chelate residual metal iron prior to the addition of Hb. The prepared media were inoculated to final OD600 = 0.005. The inocula consisted of cells grown overnight at 37°C on blood agar and suspended in iron-free CDM. Bacterial suspensions at OD600 = 0.5 were diluted 1:100 into the corresponding medium, which was then dispensed in 200 µl triplicates in a 96-well microplate (Costar 3595, Corning) at 37°C for 24 h. The experiments were performed in triplicates and were performed at least twice for each strain. Kanamycin (150 µg ml−1) was added to the medium for the growth of the complemented strain ZE4924 and ZE4935.
In silico analysis
The following accession numbers were utilized for the NEAT domain-containing proteins examined in this study: S. aureus IsdA ABX29083, S. aureus IsdC ABX29084, S. aureus IsdB YP_001332074, S. aureus IsdH Q6G8J7, S. equi ssp. zooepidemicus YP_002122760, C. novyi NT YP_877540, S. dysgalactiae ssp. equisimilis YP_002997560, S. pyogenes Shr MGAS5005 ABW80932. Identification of all protein domains was conducted by SMART analysis except the EF-hand domain, which was identified by PROSITE. Multiple sequence alignment the NEAT domains were executed using the clustalw program.
This work was supported by a grant from NIAID/NIH to Z. Eichenbaum (AI057877). We thank You Zhuo for assistance with experiments and Yu Cao for assistance with the analysis. We thank Rebecca Gunter for assistance in the construction of ZE4935 and analysis of shr mutants.