Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus

The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems.     

Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus

In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906-909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 --> NEAT-A --> NEAT-C --> IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 --> NEAT-A --> NEAT-C --> IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place.     

Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme

Cell-wall sorted proteins of the Staphylococcus aureus iron-regulated surface determinant system bind human hemoproteins, remove the heme molecule, and transport heme through the cell wall and plasma membrane for accumulation in the bacterial cytoplasm. Once inside the cell, the porphyrin ring of heme is degraded by heme degrading monooxygenases, leading to the formation of free iron for use by the bacterium as a nutrient source.     

The iron-regulated surface proteins IsdA, IsdB, and IsdH are not required for heme iron utilization in Staphylococcus aureus

The iron-regulated surface determinant proteins (Isd) of Staphylococcus aureus are expressed during iron limitation and have been proposed to be involved in the scavenging of iron from heme. In this study, the genes encoding the surface proteins IsdA, IsdB, and IsdH were inactivated in order to determine their combined role. The triple mutant was found to have no defect in growth under any conditions of iron limitation tested. Also using a mouse septic arthritis model of S.?aureus systemic disease, no significant difference in bacterial load was observed for the triple mutant, compared with its otherwise isogenic parent.     

Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdH(N1), IsdH(N2), and IsdH(N3). Here we show that they have different functions; IsdH(N1) binds Hb and Hp, whereas IsdH(N3) captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdH(N1). High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the beta-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.     

Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus

The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including Staphylococcus aureus, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an S. aureus genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the cbr locus of the plant pathogen Erwinia chrysanthemi. The first gene in the locus, which we have designated sir for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in Escherichia coli. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the S. aureus culture medium. sir-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents.     

Staphylococcus aureus determinants for nasal colonization

Approximately 20% of the healthy human population is persistently colonized in the nasal cavity with Staphylococcus aureus, which constitutes a major risk for infection. S. aureus seems to predominantly colonize the anterior part of the nasal cavity by adhering to nasal surface structures and escaping the host innate and adaptive immune responses. Several bacterial and host factors that play a role in these processes have been identified in the past few years and were in part functionally evaluated in appropriate colonization models. However, the dynamics of host-pathogen crosstalk is only partially understood.     

Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuC, fhuB, and fhuD, was identified in Staphylococcus aureus. Whereas the fhuC gene encodes an ATP-binding protein, fhuB and fhuD code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A fhuB knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.     

Metabolic flux of extracellular heme uptake in Pseudomonas aeruginosa is driven by the iron-regulated heme oxygenase (HemO)

Heme utilization by Pseudomonas aeruginosa involves several proteins required for internalization and degradation of heme. In the following report we provide the first direct in vivo evidence for the specific degradation of extracellular heme to biliverdin (BV) by the iron-regulated HemO. Moreover, through isotopic labeling ((13)C-heme) and electrospray ionization-MS analysis we have confirmed the regioselectivity and ratio of (13)C-δ and β-BV IX (70:30) is identical in vivo to that previously observed for the purified protein. Furthermore, the (13)C-BV IXδ and BV IXβ products are effluxed from the cell by an as yet unidentified transporter. Conversion of extracellular heme to BV is dependent solely on the iron-regulated HemO as evidenced by the lack of BV production in the P. aeruginosa hemO deletion strain. Complementation of P. aeruginosa ΔhemO with a plasmid expressing either the wild type HemO or α-regioselective HemO mutant restored extracellular heme uptake and degradation. In contrast deletion of the gene encoding the cytoplasmic heme-binding protein, PhuS, homologs of which have been proposed to be heme oxygenases, did not eliminate (13)C-BV IXδ and IXβ production. In conclusion the metabolic flux of extracellular heme as a source of iron is driven by the catalytic action of HemO.     

Crystal structure of the heme-IsdC complex, the central conduit of the Isd iron/heme uptake system in Staphylococcus aureus

Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the beta-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.     

Signaling and DNA-binding activities of the Staphylococcus aureus HssR-HssS two-component system required for heme sensing

For the important human pathogen Staphylococcus aureus, host heme is a vital source of nutrient iron during infection. Paradoxically, heme is also toxic at high concentrations and is capable of killing S. aureus. To maintain cellular heme homeostasis, S. aureus employs the coordinated actions of the heme sensing two-component system (HssRS) and the heme regulated transporter efflux pump (HrtAB). HssRS-dependent expression of HrtAB results in the alleviation of heme toxicity and tempered staphylococcal virulence. Although genetic experiments have defined the role of HssRS in the heme-dependent activation of hrtAB, the mechanism of this activation is not known. Furthermore, the global effect of HssRS on S. aureus gene expression has not been evaluated. Herein, we combine multivariable difference gel electrophoresis with mass spectrometry to identify the heme-induced cytoplasmic HssRS regulon. These experiments establish hrtAB as the major target of activation by HssRS in S. aureus. In addition, we show that signaling between the sensor histidine kinase HssS and the response regulator HssR is necessary for growth of S. aureus in high concentrations of heme. Finally, we show that a direct repeat DNA sequence within the hrtAB promoter is required for heme-induced, HssR-dependent expression driven by this promoter and that phosphorylated HssR binds to this direct repeat upon exposure of S. aureus to high concentrations of heme. Taken together, these data establish the mechanism for HssRS-dependent expression of HrtAB and, in turn, provide a functional understanding for how S. aureus avoids heme-mediated toxicity.     

Heme binding to the IsdE(M78A; H229A) double mutant: challenging unidirectional heme transfer in the iron-regulated surface determinant protein heme transfer pathway of Staphylococcus aureus

The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The cell-wall- and cell-membrane-associated iron-regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG, and IsdI) allow S. aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, IsdE chaperones heme to the ATP-binding-cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA, and IsdC contain at least one heme-binding near transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr-proximal amino acid)?→?IsdC-NEAT (Tyr)?→?IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. To challenge and probe this unusual unidirectional mechanism, the double mutant IsdE(M78A; H229A)-IsdE(MH)-was constructed and used in studies of heme transfer between IsdA-NEAT, IsdC-NEAT, and IsdE. This study probed the specific requirements in the heme binding site that enforce the unidirectional property of the system. Significantly, heme transfer from holo-IsdE(MH) to apo-IsdA-NEAT now occurs, breaking the established mechanism. The unique unidirectional heme-transfer properties now function under an affinity-driven mechanism. Overall, the heme proximal and distal ligands must play a crucial role controlling a gate that stops heme transfer between the native IsdE and IsdA-NEAT. We propose that these amino acids are the key control elements in the specific unidirectional protein-protein-gated release mechanism exhibited by the Isd system.     

IsdA of Staphylococcus aureus is a broad spectrum, iron-regulated adhesin

As an important facet of host-pathogen interaction, Staphylococcus aureus has the ability to adhere to human extracellular matrix (ECM) components via a range of surface proteins. Here we have shown that IsdA has broad-spectrum ligand-binding activity, including fibrinogen and fibronectin. Mapping studies revealed a distinct domain responsible for ligand binding. This domain is present in a number of iron-regulated proteins of S. aureus and in other Gram-positive organisms. The isdA gene is only expressed in iron-limited conditions under the control of Fur and not in standard laboratory media. Such conditions occur in serum in vitro and during infection. Whole cell binding and clumping assays revealed that when the bacteria are grown under iron-limited conditions, IsdA constitutes a physiologically relevant adhesin to both fibrinogen and fibronectin. Thus for S. aureus, iron is an important marker for the host environment, to which the bacterium responds by differential regulation of at least one element of its adhesive strategy.     

Characterization of the heme binding properties of Staphylococcus aureus IsdA

We report the first characterization of the physical and spectroscopic properties of the Staphylococcus aureus heme-binding protein IsdA. In this study, a combination of gel filtration chromatography and analytical centrifugation experiments demonstrate that IsdA, in solution, is a monomer and adopts an extended conformation that would suggest that it has the ability to protrude from the staphylococcal cell wall and interact with the extracellular environment. IsdA efficiently scavenged intracellular heme within Escherichia coli. Gel filtration chromatography and electrospray mass spectrometry together showed that rIsdA in solution is a monomer, and each monomer binds a single heme. Magnetic circular dichroism analyses demonstrate that the heme in rIsdA is a five-coordinate high-spin ferric heme molecule, proximally coordinated by a tyrosyl residue in a cavity that restricts access to small ligands. The heme binding is unlike that in a typical heme protein, for example, myoglobin, because we report that no additional axial ligation is possible in the high-spin ferric state of IsdA. However, reduction to ferrous heme is possible which then allows CO to axially ligate to the ferrous iron. Reoxidation forms the ferric heme, which is once again isolated from exogenous ligands. In summary, rIsdA binds a five-coordinate, high-spin ferric heme which is proximally coordinated by tyrosine. Reduction results in formation of five-coordinate, high-spin ferrous heme with a neutral axial ligand, most likely a histidine. Subsequent addition of CO results in a six-coordinate low-spin ferrous heme also with histidine likely bound proximally. Reoxidation returns the tyrosine as the proximal ligand.     

In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins.     

A Staphylococcus aureus regulatory system that responds to host heme and modulates virulence

Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality, exists as a harmless commensal in approximately 25% of humans. Identifying the molecular machinery activated upon infection is central to understanding staphylococcal pathogenesis. We describe the heme sensor system (HssRS) that responds to heme exposure and activates expression of the heme-regulated transporter (HrtAB). Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. We suggest that the coordinated activity of Hss and Hrt allows S. aureus to sense internal host tissues, resulting in tempered virulence to avoid excessive host tissue damage. Further, genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory system by which Gram-positive pathogens sense heme as a molecular marker of internal host tissue and modulate virulence.     

Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells

Using human erythroleukemia K562 cells, existence of receptors for hemopexin has been investigated. Hemopexin was bound to the cells in saturable, time- and temperature-dependent manner. The cells exhibited approximately 8,400 binding sites/cell for hemopexin and apohemopexin. The dissociation constants (Kd) for hemopexin and apohemopexin were 4.79 nM and 10.8 nM, respectively. Specific binding of labeled hemopexin was inhibited with increasing concentrations of unlabeled hemopexin and apohemopexin, but unaffected by transferrin and serum albumin. Heme bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that heme in hemopexin was taken up by K562 cells via the receptors for hemopexin.