9.A.39 The Gram-positive Bacterial Hemoglobin Receptor (Isd) Family
The pathogenesis of human infections caused by the gram-positive microbe Staphylococcus aureus is reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing four cell wall-anchored proteins (IsdA, IsdB, IsdC and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Torres et al. (2006) reported that S. aureus can utilize the host hemoproteins, hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. They demonstrated that staphylococci capture hemoglobin on the bacterial surface via IsdB, and that inactivation of isdB, but not isdA or isdH, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of S. aureus to utilize hemoglobin as an iron source. IsdB removes heme iron from hemoglobin and transports it into the cell (Torres et al., 2006). Liu et al. (2008) showed that hemin uptake involves transfer from IsdA to IsdC on the cell surface.
IsdB contains two N- and C-terminal peaks of striking hydrophobicity and two central Near Iron Transporter (NEAT) domains (residues 150-260 and 370-450). This domain is involved in iron transport, sometimes complexed with a siderophore. It has an N-terminal YSIRK type signal peptide which contains the conserved motif: [YF]SIRK X3 GX2S [VIA] and a C-terminal Gram-positive anchor domain with the LPXTG sortase processing site. IsdA,B,C,D and H all have an N-terminal peak of hydrophobicity and C-terminal peak of hydrophobicity, and they all may contain NEAT domains.
NEAT domains in IsdH/HarA extract heme from methemoglobin (Pilpa et al., 2009). IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a Near Iron Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX) (Villareal et al., 2008). IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its β-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. IsdC weakly binds heme (K(D) = 0.34 %u03BCM, and ZnPPIX rapidly dissociates from the protein. NMR studies of the apo-form of IsdC reveal that a 3(10) helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins (Villareal et al., 2008).
All of the close homologues of IsdB occur in low G C Gram-positive bacteria. These include the haptoglobin-binding surface anchored proteins of S. aureus (BAF67896; 895 aas, 39% identity). However more distant homologues with overlapping domains are found in eukaryotes. Grigg et al. (2010) have reviewed the Isd system of S. aureus.
B. anthracis, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Tarlovsky et al. (2010) presented evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection.
Bacillus anthracis secretes two NEAT (near iron transporter) proteins, IsdX1 and IsdX2, which scavenge heme from host hemoglobin and promote growth under low iron conditions. Fabian et al. (2009) presented evidence that the heme-bound form of IsdX1 rapidly and directionally transfers heme to IsdC, a NEAT protein covalently attached to the cell wall. The transfer of heme is mediated by a physical association between the donor and recipient. Unlike Staphylococcus aureus, whose NEAT proteins acquire heme from hemoglobin directly at the bacterial surface, B. anthracis secretes IsdX1 to capture heme in the extracellular milieu and relies on NEAT-NEAT interactions to deliver the bound heme to the envelope via IsdC.
The pathway for iron utilization in S. aureus appears to be:
substrate iron containing protein --> Receptors (IsdH, A, B) --> Cell wall conduit (IsdC) --> Membrane ABC transporter (IsdDEF) --> Cytoplasmic heme deregulation and iron release (IsdGI)
Iron regulated surface determinant, Haptoglobin receptor A (HarA or IsdH) precursor (891 aas). Also binds hemoglobin and haptoglobin/hemoglobin complexes with nM or µM binding constants to an 8-stranded anti-parallel β-barrel domain (Dryla et al., 2007). The functionally distinct NEAT (NEAr Transporter) domain within IsdH extracts heme from methemoglobin (Pilpa et al., 2009). Hemoglobin/myoglobin receptor B (IsdB), transferrin, hemin, fetuin, asialofetuin receptor (IsdA), and the cell wall conduit, IsdC may comprise the full system (Villareal et al., 2008).
IsdHABC of Staphylococcus aureus
IsdH (HarA) (Q931P4)
Iron-regulated surface determinant, IsdA (IsdA binds transferrin, heme, hemoglobin, hemin, fetuin, asialo fetuin, protein A, fibronectin and fibrinogen) (Liu et al., 2008). The pathway for uptake of heme from human methemoglobin by the iron-regulated surface determinants (Isd) is: B (9.A.39.1.1)→ A (9.A.39.1.3) → C (9.A.39.1.4) → E (3.A.1.14.18)→ DF (3.A.1.14.18) → cytoplasm (Zhu et al., 2008).
IsdA of Staphylococcus aureus (354aas; 2TMSs at the N- and C-termini) (P0C1S5)
Iron-regulated surface determinant, IsdC (interacts functionally with IsdA; Liu et al., 2008). The path way for uptake of heme from human methemoglobin by the iron-regulated surface determinants (Isd) is: B(9.A.39.1.1)→ A(9.A.39.1.3)→ C(9.A.39.1.4)→ E(3.A.1.14.18)→ DF(3.A.1.14.18)→ cytoplasm (Zhu et al., 2008). (227aas; 2TMSs at the N- and C- termini)
IsdC of Staphylococcus aureus (Q8KQR1)
Iron-regulated surface determinant (Isd) system: S-layer homologue domain protein (SHDP). IsdX1 and IsdX2 are hemophores that feed heme to IsdC surface protein (Fabian et al., 2009; Tarlovsky et al., 2010).
The IsdC/SHDP iron transporter of Bacillus anthracis
SKN1 domain-containing protein of 364 aas and 2 TMSs
SKN1 protein of Leuconostoc carnosum