Structure and role of the linker domain of the iron surface-determinant protein IsdH in heme transportation in Staphylococcus aureus.

Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAr-iron Transporter (NEAT) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route toward IsdH do not play a critical role in the transfer rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor.

BACKGROUND: For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood. METHODS: An alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques. RESULTS: HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand. CONCLUSION: In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter. GENERAL SIGNIFICANCE: A detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.

Loss of complex-type N-linked glycans attenuates maximum cell density and susceptibility to human serum of Trypanosoma brucei brucei.

Trypanosoma brucei brucei is a parasitic protist that expresses cell surface proteins modified with complex-type N-linked glycan (NLG), like multicellular organisms. However, little is known about the role of complex-type NLG. In T. b. brucei, it has been shown that either one of the glycosyltransferases, TbGT11 or TbGT15, is sufficient to initiate the synthesis of complex-type NLG. To clarify the role of complex-type NLG, it is necessary to generate cells lacking both enzymes. Therefore, we deleted TbGT11 and TbGT15 from the genome of T. b. brucei for the phenotypic examination. The mutant strain grew in culture, with reduced maximum cell density; showed decreased susceptibility to normal human serum, which contains trypanolytic factors; and lacked uptake of the haptoglobin-hemoglobin complex. These data indicate that protein modification by complex-type NLG is not essential but is required for receptor function.

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 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.

Molecular mechanisms of Porphyromonas gingivalis-host cell interaction on periodontal diseases.

Porphyromonas gingivalis (P. gingivalis) is a major oral pathogen and associated with periodontal diseases including periodontitis and alveolar bone loss. In this review, we indicate that two virulence factors, which are hemoglobin receptor protein (HbR) and cysteine proteases "gingipains", expressed by P. gingivalis have novel functions on the pathogenicity of P. gingivalis. P. gingivalis produces three types of gingipains and concomitantly several adhesin domains. Among the adhesin domains, hemoglobin receptor protein (HbR), also called HGP15, has the function of induction of interleukin-8 (IL-8) expression in human gingival epithelial cells, indicating the possibility that HbR is associated with P. gingivalis-induced periodontal inflammation. On bacteria-host cells contact, P. gingivalis induces cellular signaling alteration in host cells. Phosphatidylinositol 3-kinase (PI3K) and Akt are well known to play a pivotal role in various cellular physiological functions including cell survival and glucose metabolism in mammalian cells. Recently, we demonstrated that gingipains attenuate the activity of PI3K and Akt, which might have a causal influence on periodontal diseases by chronic infection to the host cells from the speculation of molecular analysis. In this review, we discuss new molecular and biological characterization of the virulence factors from P. gingivalis.

Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense.

BACKGROUND: Since Trypanosoma spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life-cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T. brucei Hp-Hb complex receptor (TbHpHbR). METHODS: Trypanosoma congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. RESULTS: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K d) = 2.1×10(-8) M) but free-Hp with low affinity (K d = 2.2×10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). CONCLUSION: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T. congolense EMF-specific Hb receptor (TcEpHbR).

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.