Directed Inter-domain Motions Enable the IsdH Staphylococcus aureus Receptor to Rapidly Extract Heme from Human Hemoglobin.

Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tri-domain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globin's heme pocket. Using molecular simulations coupled with Markov modeling, along with stopped-flow experiments to quantitatively measure heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hb's pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

The Role of Iron in Staphylococcus aureus Infection and Human Disease: A Metal Tug of War at the Host-Microbe Interface.

Iron deficiency anemia can be treated with oral or intravenous Fe supplementation. Such supplementation has considerable effects on the human microbiome, and on opportunistic pathogenic micro-organisms. Molecular understanding of the control and regulation of Fe availability at the host-microbe interface is crucial to interpreting the side effects of Fe supplementation. Here, we provide a concise overview of the regulation of Fe by the opportunistic pathogen Staphylococcus aureus. Ferric uptake regulator (Fur) plays a central role in controlling Fe uptake, utilization and storage in order to maintain a required value. The micro-organism has a strong preference for heme iron as an Fe source, which is enabled by the Iron-regulated surface determinant (Isd) system. The strategies it employs to overcome Fe restriction imposed by the host include: hijacking host proteins, replacing metal cofactors, and replacing functions by non-metal dependent enzymes. We propose that integrated omics approaches, which include metalloproteomics, are necessary to provide a comprehensive understanding of the metal tug of war at the host-microbe interface down to the molecular level.

Simultaneous Exposure to Intracellular and Extracellular Photosensitizers for the Treatment of Staphylococcus aureus Infections.

Staphylococcus aureus is a serious threat to public health due to the rise of antibiotic resistance in this organism, which can prolong or exacerbate skin and soft tissue infections (SSTIs). Methicillin-resistant S. aureus is a Gram-positive bacterium and a leading cause of SSTIs. As such, many efforts are under way to develop therapies that target essential biological processes in S. aureus. Antimicrobial photodynamic therapy is an effective alternative to antibiotics; therefore we developed an approach to simultaneously expose S. aureus to intracellular and extracellular photosensitizers. A near infrared photosensitizer was conjugated to human monoclonal antibodies (MAbs) that target the S. aureus iron-regulated surface determinant (Isd) heme acquisition proteins. In addition, the compound VU0038882 was developed to increase photoactivatable porphyrins within the cell. Combinatorial photodynamic treatment of drug-resistant S. aureus exposed to VU0038882 and conjugated anti-Isd MAbs proved to be an effective antibacterial strategy in vitro and in a murine model of SSTIs.

NEAr Transporter (NEAT) Domains: Unique Surface Displayed Heme Chaperones That Enable Gram-Positive Bacteria to Capture Heme-Iron From Hemoglobin.

Iron is an important micronutrient that is required by bacteria to proliferate and to cause disease. Many bacterial pathogens forage iron from human hemoglobin (Hb) during infections, which contains this metal within heme (iron-protoporphyrin IX). Several clinically important pathogenic species within the Firmicutes phylum scavenge heme using surface-displayed or secreted NEAr Transporter (NEAT) domains. In this review, we discuss how these versatile proteins function in the Staphylococcus aureus Iron-regulated surface determinant system that scavenges heme-iron from Hb. S. aureus NEAT domains function as either Hb receptors or as heme-binding chaperones. In vitro studies have shown that heme-binding NEAT domains can rapidly exchange heme amongst one another via transiently forming transfer complexes, leading to the interesting hypothesis that they may form a protein-wire within the peptidoglycan layer through which heme flows from the microbial surface to the membrane. In Hb receptors, recent studies have revealed how dedicated heme- and Hb-binding NEAT domains function synergistically to extract Hb's heme molecules, and how receptor binding to the Hb-haptoglobin complex may block its clearance by macrophages, prolonging microbial access to Hb's iron. The functions of NEAT domains in other Gram-positive bacteria are also reviewed.

The Staphylococcus aureus IsdH Receptor Forms a Dynamic Complex with Human Hemoglobin that Triggers Heme Release via Two Distinct Hot Spots.

Iron is an essential nutrient that is actively acquired by bacterial pathogens during infections. Clinically important Staphylococcus aureus obtains iron by extracting heme from hemoglobin (Hb) using the closely related IsdB and IsdH surface receptors. In IsdH, extraction is mediated by a conserved tridomain unit that contains its second (N2) and third (N3) NEAT domains joined by a helical linker, called IsdHN2N3. Leveraging the crystal structure of the IsdHN2N3:Hb complex, we have probed the mechanism of heme capture using NMR, stopped-flow transfer kinetics measurements, and molecular dynamics (MD) simulations. NMR studies of the 220 kDa IsdHN2N3:Hb complex reveal that it is dynamic, with persistent interdomain motions enabling the linker and N3 domains in the receptor to transiently engage Hb to remove its heme. An alanine mutagenesis analysis reveals that two receptor subsites positioned ~20 Å apart trigger heme release by contacting Hb's F-helix. These subsites are located within the N3 and linker domains and appear to play distinct roles in stabilizing the heme transfer transition state. Linker domain contacts primarily function to destabilize Hb-heme interactions, thereby lowering ΔH‡, while contacts from the N3 subsite play a similar destabilizing role, but also form a bridge through which heme moves from Hb to the receptor. Interestingly, MD simulations suggest that within the transiently forming interface, both the F-helix and receptor bridge are in motion, dynamically sampling conformations that are suitable for heme transfer. Thus, IsdH triggers heme release from Hb via a flexible, low-affinity interface that forms fleetingly in solution.

Human mAbs to Staphylococcus aureus IsdA Provide Protection Through Both Heme-Blocking and Fc-Mediated Mechanisms.

The nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.

Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus.

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It actively acquires the essential nutrient iron from human hemoglobin (Hb) using the iron-regulated surface-determinant (Isd) system. This process is initiated when the closely related bacterial IsdB and IsdH receptors bind to Hb and extract its hemin through a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. Previously, we demonstrated that the tri-domain unit within IsdH (IsdHN2N3) triggers hemin release by distorting Hb's F-helix. Here, we report that IsdHN2N3 promotes hemin release from both the α- and ß-subunits. Using a receptor mutant that only binds to the α-subunit of Hb and a stopped-flow transfer assay, we determined the energetics and micro-rate constants of hemin extraction from tetrameric Hb. We found that at 37 °C, the receptor accelerates hemin release from Hb up to 13,400-fold, with an activation enthalpy of 19.5 ± 1.1 kcal/mol. We propose that hemin removal requires the rate-limiting hydrolytic cleavage of the axial HisF8 Nϵ-Fe3+ bond, which, based on molecular dynamics simulations, may be facilitated by receptor-induced bond hydration. Isothermal titration calorimetry experiments revealed that two distinct IsdHN2N3·Hb protein·protein interfaces promote hemin release. A high-affinity receptor·Hb(A-helix) interface contributed ∼95% of the total binding standard free energy, enabling much weaker receptor interactions with Hb's F-helix that distort its hemin pocket and cause unfavorable changes in the binding enthalpy. We present a model indicating that receptor-introduced structural distortions and increased solvation underlie the IsdH-mediated hemin extraction mechanism.

The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin.

Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.

IsdB-dependent hemoglobin binding is required for acquisition of heme by Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogen responsible for tremendous morbidity and mortality. As with most bacteria, S. aureus requires iron to cause disease, and it can acquire iron from host hemoglobin. The current model for staphylococcal hemoglobin-iron acquisition proposes that S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB. IsdB removes heme from bound hemoglobin and transfers this cofactor to other proteins of the Isd system, which import and degrade heme to release iron in the cytoplasm. Here we demonstrate that the individual components of the Isd system are required for growth on low nanomolar concentrations of hemoglobin as a sole source of iron. An in-depth study of hemoglobin binding by IsdB revealed key residues that are required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a sole iron source. These processes are found to contribute to the pathogenicity of S. aureus in a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis of S. aureus infection.