Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

The streptococcal iron uptake (Siu) transporter is required for iron uptake and virulence in a zebrafish infection model.

A limited understanding of iron uptake mechanisms is available for Streptococcus pyogenes, a haemolytic human pathogen capable of using a variety of haemoproteins in addition to ferric and ferrous iron. This study characterizes a transporter named siu (for streptococcal iron uptake), which consists of an ATP-binding protein (SiuA), a substrate-binding protein (SiuD), and two membrane permease subunits (SiuBG). An siuG mutant was constructed and characterized. The mutant demonstrated growth reduction in comparison to the parent strain when grown in complex medium containing iron in the form of blood, haemoglobin or serum. Only a small reduction in the growth of the siuG mutant was observed in medium containing ferric iron. However, in iron uptake assays the siuG mutant showed a decrease of approximately 30 % in Fe3+ incorporation. Addition of 6 microM haem to the medium inhibited Fe3+ uptake by the wild-type by 76 %, while addition of protoporphyrin IX did not, suggesting that utilization of haem as an iron source is responsible for the inhibition of Fe3+ uptake. Inactivation of siuG moderately reduced the ability of haem to inhibit Fe3+ incorporation by the cells. Inactivation of siaB (encoding a membrane permease of a second iron transporter) had a similar outcome, and inactivation of both transporters had a cumulative effect. These observations implicate both the siu and sia transporters in haem utilization by Strep. pyogenes. Studies in a zebrafish infection model revealed that the siuG mutant was attenuated in both intramuscular and intraperitoneal routes of infection. Together these observations show that the siu system is an iron acquisition pathway in Strep. pyogenes that is important both in vitro and in vivo.

Identification and characterization of a Streptococcus pyogenes operon involved in binding of hemoproteins and acquisition of iron.

The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.