Sensing the host environment: recognition of hemoglobin by the pathogenic yeast Candida albicans

Adhesion to host cells and tissues is important for several steps in the pathogenesis of disseminated Candida albicans infections. Although such adhesion is evident in vivo and for C. albicans grown in vitro in complex medium, some adhesive activities are absent when cultures are grown in defined media. However, addition of hemoglobin to defined media restores binding and adhesion to several host proteins. This activity of hemoglobin is independent of iron acquisition and is mediated by a cell surface hemoglobin receptor. In addition to regulating expression of adhesion receptors, hemoglobin rapidly induces expression of several genes. One of these, a heme oxygenase, allows the pathogen to utilize exogenous heme or hemoglobin to acquire iron and to produce the cytoprotective molecules alpha-biliverdin and carbon monoxide. The specific recognition of and responses to hemoglobin demonstrate a unique adaptation of C. albicans to be both a commensal and an opportunistic pathogen in humans.     

Point mutations in HpuB enable gonococcal HpuA deletion mutants to grow on hemoglobin.

Neisseria gonorrhoeae ordinarily requires both HpuA and HpuB to use hemoglobin (Hb) as a source of iron for growth. Deletion of HpuA resulted in reduced Hb binding and failure of growth on Hb. We identified rare Hb-utilizing colonies (Hb(+)) from an hpuA deletion mutant of FA1090, which fell into two phenotypic classes. One class of the Hb(+) revertants required expression of both TonB and HpuB for growth on Hb, while the other class required neither TonB nor HpuB. All TonB/HpuB-dependent mutants had single amino acid alterations in HpuB, which occurred in clusters, particularly near the C terminus. The point mutations in HpuB did not restore normal Hb binding. Human serum albumin inhibited Hb-dependent growth of HpuB point mutants lacking HpuA but did not inhibit growth when expression of HpuA was restored. Thus, HpuB point mutants internalized heme in the absence of HpuA despite reduced binding of Hb. HpuA facilitated Hb binding and was important in allowing use of heme from Hb for growth.     

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.     

Staphylococcus aureus Uses a Novel Multidomain Receptor to Break Apart Human Hemoglobin and Steal Its Heme

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdH^N2N3, Ala³²⁶–Asp⁶⁶⁰) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdH^N2N3 extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.     

Structure of the Hemoglobin-IsdH Complex Reveals the Molecular Basis of Iron Capture by Staphylococcus aureus

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and β Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/β globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.     

Selective binding of antimicrobial porphyrins to the heme‐receptor IsdH‐NEAT3 of Staphylococcus aureus

The Isd (iron‐regulated surface determinant) system of the human pathogen Staphylococcus aureus is responsible for the acquisition of heme from the host organism. We recently reported that the extracellular heme receptor IsdH‐NEAT3 captures and transfers noniron antimicrobial porphyrins containing metals in oxidation state (III). However, it is unclear if geometric factors such as the size of the metal (ionic radius) affect binding and transfer of metalloporphyrins. We carried out an ample structural, functional, and thermodynamic analysis of the binding properties of antimicrobial indium(III)‐porphyrin, which bears a much larger metal ion than the iron(III) of the natural ligand heme. The results demonstrate that the NEAT3 receptor recognizes the In(III)‐containing PPIX in a manner very similar to that of heme. Site‐directed mutagenesis identifies Tyr642 as the central element in the recognition mechanism as suggested from the crystal structures. Importantly, the NEAT3 receptor possesses the remarkable ability to capture dimers of metalloporphyrin. Molecular dynamics simulations reveal that IsdH‐NEAT3 does not require conformational changes, or large rearrangements of the residues within its binding site, to accommodate the much larger (heme)₂ ligand. We discuss the implications of these findings for the design of potent inhibitors against this family of key receptors of S. aureus.     

Control of hemoglobin synthesis in erythroid differentiating K562 cells: II. Studies of iron mobilization in erythroid cells by high-performance liquid chromatography–electrochemical detection

We have demonstrated that iron controls hemoglobin (Hb) synthesis in erythroid differentiating K562 cells by enhancing the activity of a key enzyme of the Hb synthesis, δ-aminolevulinate synthase (ALAS). In the present study, we studied iron mobilization and the role of iron in erythroid differentiating cells by measuring the level of iron by means of high-performance liquid chromatography using electrochemical detection (HPLC–ED). After treatment of K562 cells with sodium butyrate, the expression of transferrin receptor (TfR) increased initially, followed by an increase in the levels of both total iron and Hb as well as the ALAS activity. However, no increase could be found in the levels of non-heme iron, low-molecular-mass iron (LMMFe) and ferritin. Addition of diferric transferrin (FeTf) enhanced both δ-aminolevulinic acid (ALA) and Hb synthesis. In contrast, addition of hemin elevated the levels of all iron species as well as the Hb synthesis but reduced the TfR expression and ALA contents in both butyrate treated and untreated cells. These results suggest that Hb synthesis is controlled by TfR expression, and that the ALA synthesis is suppressed by iron released from heme and/or Hb due to lowered expression of TfR.     

Candida albicans Als3, a multifunctional adhesin and invasin

Candida albicans is part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. For C. albicans to persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. The C. albicans hypha-specific surface protein Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii. Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Because of its multiple functions and its high expression level in vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.     

Indoleamine 2,3-dioxygenase and iron are required for Mycobacterium leprae survival

Our previous study has demonstrated that IL-10 may modulate both indoleamine 2,3-dioxygenase (IDO) and CD163 expression in lepromatous leprosy (LL) cells, favoring Mycobacterium leprae persistence through induction of regulatory pathways and iron storage. Here, we observed that in LL lesion cells there is an increase in the expression of proteins involved in iron metabolism such as hemoglobin (Hb), haptoglobin, heme oxygenase 1 and transferrin receptor 1 (TfR1) when compared to tuberculoid leprosy (BT) cells. We also found increased iron deposits and diminished expression of the iron exporter ferroportin 1 in LL lesion cells. Hemin, but not FeSO₄ stimulation, was able to enhance M. leprae viability by a mechanism that involves IDO. Analysis of cell phenotype in lesions demonstrated a predominance of M2 markers in LL when compared with BT lesion cells. A positive correlation between CD163 and PPARG with the bacillary index (BI) was observed. In contrast, TNF, STAT1 and CSF2 presented a negative correlation with the BI. In summary, this study demonstrates that iron may regulate IDO expression by a mechanism that involves IL-10, which may contribute for the predominance of M2-like phenotype in LL lesions that favors the phagocytosis and maintenance of M. leprae in host cells.     

Oxygen induction of epithelial Na(+) transport requires heme proteins

Fetal distal lung epithelial (FDLE) cells exposed to a postnatal O(2) concentration of 21% have higher epithelial Na(+) channel (ENaC) mRNA levels and Na(+) transport relative to FDLE cells grown in a fetal O(2) concentration of 3%. To investigate the mechanism of this process, FDLE monolayers were initially cultured in 3% O(2), and then some were switched to a 21% O(2) environment. Incubation of FDLE cells with the iron chelator deferoxamine, CoCl(2), NiCl(2), or an inhibitor of heme synthesis prevented or diminished the O(2) induction of amiloride-sensitive short-circuit current in FDLE cells. Similarly, defer- oxamine and cobalt prevented O(2)-induced ENaC mRNA expression. Exposure of FDLE cells grown under hypoxic conditions to carbon monoxide increased both ENaC mRNA expression and amiloride-sensitive short-circuit current. We therefore concluded that induction of ENaC mRNA expression and amiloride-sensitive Na(+) transport in FDLE cells by a physiological increase in O(2) concentration seen at birth requires iron and heme proteins.     

Extra domain A-containing fibronectin expression in Spin90-deficient fibroblasts mediates cancer-stroma interaction and promotes breast cancer progression

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4β1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4β1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4β1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.     

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.     

Fungal recognition enhances mannose receptor shedding through dectin-1 engagement

The mannose receptor (MR) is an endocytic type I membrane molecule with a broad ligand specificity that is involved in both hemostasis and pathogen recognition. Membrane-anchored MR is cleaved by a metalloproteinase into functional soluble MR (sMR) composed of the extracellular domains of intact MR. Although sMR production was initially considered a constitutive process, enhanced MR shedding has been observed in response to the fungal pathogen Pneumocystis carinii. In this work, we have investigated the mechanism mediating enhanced MR shedding in response to fungi. We show that other fungal species, including Candida albicans and Aspergillus fumigatus, together with zymosan, a preparation of the cell wall of Saccharomyces cerevisiae, mimic the effect of P. carinii on sMR production and that this effect takes place mainly through β-glucan recognition. Additionally, we demonstrate that MR cleavage in response to C. albicans and bioactive particulate β-glucan requires expression of dectin-1. Our data, obtained using specific inhibitors, are consistent with the canonical Syk-mediated pathway triggered by dectin-1 being mainly responsible for inducing MR shedding, with Raf-1 being partially involved. As in the case of steady-state conditions, MR shedding in response to C. albicans and β-glucan particles requires metalloprotease activity. The induction of MR shedding by dectin-1 has clear implications for the role of MR in fungal recognition, as sMR was previously shown to retain the ability to bind fungal pathogens and can interact with numerous host molecules, including lysosomal hydrolases. Thus, MR cleavage could also impact on the magnitude of inflammation during fungal infection.     

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O₂ or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β₄ homotetramer) and, therefore, are associated with the α–β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O₂ binding mechanism of Hb A.