INTRODUCTION

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Candida albicans is an important opportunistic pathogen for humans, causing both superficial and disseminated infections (reviewed in Refs. 1 and 2) with significant morbidity and mortality among immunocompromised patients (3). Adhesion of the organism to mucosal epithelium is a prerequisite for colonization and, therefore, is regarded as an initial step in the process leading to infections (3, 4). Additional adhesion events to endothelium and extracellular matrix (ECM)¹ components are required for dissemination of C. albicans. A number of ECM proteins bind to C. albicans, including fibronectin (5-8), laminin (9), vitronectin (10, 11), complement (12), fibrinogen (13), gelatin (7), and types I and IV collagen (14).

Interaction with individual ECM proteins is mediated by binding to respective receptors on the surface of Candida cells (5-7, 9-11). Some of these receptors are probably homologs of mammalian integrins (15). However, fibronectin binds to C. albicans both through the cell binding domain recognized by mammalian integrins (8) and through the collagen binding domain (6), and laminin and fibrinogen bind to distinct classes of receptors on C. albicans that are probably not integrins (9, 13, 16). Thus, both integrin and non-integrin receptors may mediate adhesion of C. albicans.

Previously, we reported that hemoglobin induces a marked enhancement of fibronectin binding activity in C. albicans (17). This induction is reversible, requires cell growth in the presence of hemoglobin, and is not due to a bridge effect of hemoglobin between a receptor on the organism and fibronectin. In addition, adhesion of C. albicans to corneal endothelial cells was significantly increased when grown in hemoglobin-containing defined medium compared with those grown in defined medium alone. Although the ability to acquire iron has long been considered one of the most important adaptive responses for microbial pathogenesis (18, 19), the enhancement of fibronectin binding by hemoglobin was not simply due to iron acquisition from the hemoglobin, because other ferroproteins, ferrous ions, or iron-containing porphyrins were inactive (17). Thus, hemoglobin itself may act as a potent regulator for the fibronectin receptor in C. albicans, although its precise mechanism remains unclear.

We have now examined whether this specific induction by hemoglobin influences binding of C. albicans to other ECM proteins. We demonstrate that binding of C. albicans to laminin, fibrinogen, and type IV collagen, are increased to various degrees, but binding to thrombospondin-1 and type I collagen are not. In addition to inducing specific receptors for some ECM ligands on C. albicans, growth in the presence of hemoglobin induces a promiscuous receptor for several ECM proteins including fibronectin (FN), fibrinogen, and laminin. Adhesion to these proteins is also coordinately increased, and expression of specific cell surface proteins that bind FN is increased. Among these, a 55-kDa protein was identified as a hemoglobin-induced fibronectin receptor. Based on these findings, hemoglobin seems to be a potent regulator of the adhesive phenotype of C. albicans.

	EXPERIMENTAL PROCEDURES

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Strains and Growth Conditions-- ATCC strain 44807 of C. albicans was purchased from the American Type Culture Collection and used throughout this study. The organism was initially grown in Sabouraud medium (6) to early stationary phase at 26 °C, and blastoconidia were stored at 70 °C with 20% sterile glycerol until used. On a biweekly basis, a fresh culture was made as a stock for daily use. For each experiment, the organism was inoculated into the 4 × of yeast nitrogen base (YNB) broth with or without hemoglobin at a final concentration of 0.1% and incubated at 26 °C for 20-48 h (17). YNB is a chemically defined medium, and under this growth condition no germination was found upon microscopic examination of cultures in the presence or absence of hemoglobin.

Extracellular Matrix Proteins-- FN was purified from frozen human plasma as described previously (6). Thrombospondin-1 was isolated from human platelets (20). Mouse laminin and gelatin were purchased from Life Technologies, Inc., bovine fibrinogen was from Calbiochem, and collagens type I and IV were from Collaborative Research Incorporated (Bedford, MA). Iodination of individual ECM proteins was accomplished using Iodogen (Pierce), and unbound iodine was removed by gel filtration through a PD-10 column (6).

Cell Binding Assay-- In a typical binding assay, 2 × 10⁶ C. albicans were exposed to each ¹²⁵I matrix protein at a final concentration of 0.5 µg/ml in a total volume of 200 µl of Dulbecco's phosphate-buffered saline (PBS) without CaCl₂ and MgCl₂, 0.1% BSA, pH 6.0, in 12 × 75-mm polypropylene tubes (PGC, Gaithersburg, MD) and incubated for 3 h at room temperature with shaking at 160 rpm. The cell suspensions were then transferred to microfuge tubes, and the blastoconidia were separated from unbound radioactive ligand by centrifugation through 100 µl of an oil mixture of dibutyl phthalate/dioctyl phthalate (2:1). Radioactive ECM protein bound to the cell pellet was quantified in a gamma counter (Packard Instrument Company, Downers Grove, IL). For inhibition assays, the binding of radiolabeled protein was determined in the presence of various concentrations of unlabeled homologous or heterologous ECM ligands.

Adhesion to Immobilized Matrix Proteins-- Extracellular matrix proteins were coated onto glass chamber slides (Nalge Nunc International, Naperville, IL) wells by adding 300 µl/well of FN solution at 1 or 10 µg/ml into each well and incubating at 4 °C overnight. Adhesion was measured as described previously (17).

Biotinylation and Extraction of Surface Proteins of C. albicans-- Biotinylation of Candida surface proteins was achieved using sulfohydroxysuccinimidyl-6-(biotinamido) hexanoate (Pierce). Briefly, 20 ml of Candida cultures grown in 4 × YNB with or without hemoglobin at 26 °C for 48 h were harvested by centrifugation, and the cells were washed twice with DPBS, pH 7.4, without Ca²⁺ and Mg²⁺ (Life Technologies, Inc.) to remove trapped or precipitated hemoglobin. The pellets were suspended in 1 ml of 50 mM sodium bicarbonate buffer, pH 8.5, in microfuge tubes, and 74 µl of 1 mg/ml sulfohydroxysuccinimidyl-6-(biotinamido) hexanoate were added. The cell suspension was incubated at room temperature with shaking for 1 h, and excess biotinylation reagent was removed by washing the cells with DPBS. After biotinylation, cell surface proteins were extracted. Candida cells were suspended either in lyticase (Sigma) at a final concentration of 2 mg/ml, 8 mM dithiothreitol, or a combination of both and incubated at 37 °C for 1 h with vigorous shaking. Cell pellets were removed by centrifugation, and extracted cell surface proteins in the supernatant fluids were either separated by 10% SDS-polyacrylamide gel electrophoresis containing 8 M urea or stored at 70 °C for future use. After electrophoresis, proteins were transferred onto a nitrocellulose membrane, blocked with 3% BSA in 50 mM Tris, pH 7.5, 150 mM NaCl, and incubated with streptavidin-horseradish peroxidase (Amersham Life Science, Inc.). Biotinylated cell surface proteins were visualized using an ECL chemiluminescent detection kit (Amersham).

Purification of a FN-binding Protein Using Affinity Chromatography-- For preparation of the affinity gel, FN was conjugated to Reacti-Gel (Pierce) at a concentration of 1 mg/ml at 4 °C overnight, and excess FN was removed by washing. Unbound reactive groups were blocked by incubating with 100 mM Tris buffer, pH 7.5, at room temperature for 1 h. Extracted Candida biotinylated surface proteins were incubated with a suspension of the affinity gel at room temperature for 4 h and transferred to a glass column. The column was washed with 10 column volumes of DPBS, pH 7.4, containing 300 mM NaCl. Bound proteins were eluted stepwise with DPBS buffer, pH 7.4, containing 650 mM NaCl followed by 0.2 M sodium acetate, pH 4.0. Eluted fractions were dialyzed, freeze-dried, and analyzed by SDS-polyacrylamide gel electrophoresis. FN-binding cell surface proteins were visualized by ECL detection.

	RESULTS

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Induction of ECM Protein Binding by Hemoglobin Is Not Limited to Fibronectin-- After growth in YNB supplemented with 0.1% hemoglobin for 48 h, binding of Candida cells to several radiolabeled ECM proteins was examined. Among the ECM proteins tested, binding of fibronectin, laminin, fibrinogen, and type IV collagen increased more than 10-fold relative to their binding to cells grown in the absence of hemoglobin (Fig. 1). In contrast, hemoglobin failed to enhance the binding of type I collagen or thrombospondin-1 to C. albicans. Binding of FN, laminin, fibrinogen, and type IV collagen were consistently and markedly enhanced in all experiments. Some enhancement of gelatin binding was observed but varied from 2 to 16-fold in three separate experiments (results not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of hemoglobin supplement in yeast nitrogen base medium on extracellular matrix protein binding to C. albicans. Candida cells were inoculated into 4 × YNB defined medium with or without hemoglobin at a final concentration of 0.1% and incubated at room temperature for 48 h on a rocking platform. Binding activity of each protein was determined as described under "Experimental Procedures" and is expressed as the -fold increase between cultures grown in the presence of hemoglobin and in the absence of hemoglobin. LN, laminin; FG, fibrinogen; T-IV, type IV collagen; T-I, type I collagen; and TSP, thrombospondin-1. Results are presented as mean ± S.D., n = 3.

Hemoglobin Enhances Adhesion of Candida albicans to Several Immobilized Ligands-- Although increased binding of soluble matrix proteins could have biological significance, the ability of immobilized ECM proteins to promote C. albicans adhesion to a surface is the parameter that is required for dissemination of the pathogen. We previously reported that hemoglobin induced an increased adhesion of C. albicans to immobilized FN coated onto glass Chamber slides (17). Using the same technique, adhesion of C. albicans grown in hemoglobin-containing medium was examined on slides coated with various ECM proteins (Fig. 2). Compared with the cells grown in the absence of hemoglobin, hemoglobin-induced C. albicans demonstrated a significantly greater adhesion (p < 0.05 by a two-tailed t test) to immobilized FN, laminin, fibrinogen, or type IV collagen at a concentration of 10 µg/ml, respectively. Although adhesion to type I collagen increased slightly for Candida cells prepared from cultures grown in the presence of hemoglobin, the difference was not statistically significant (p > 0.1). Growth in medium containing hemoglobin did not alter adhesion of C. albicans to immobilized thrombospondin-1 or gelatin (p > 0.2).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Growth of C. albicans in hemoglobin enhances adhesion to immobilized ECM proteins. Each ECM protein was adsorbed at 10 µg/ml in Dulbecco's PBS without Ca2+ and Mg2+, pH 7.4, to Chamber slides by incubation overnight at 4 °C. Unbound protein was removed by washing three times with Dulbecco's PBS. Candida cells prepared from cultures with (solid bars) or without (striped bars) hemoglobin in the YNB medium were added to each well at a concentration of 2 × 106 colony-forming units/ml and allowed to incubate at room temperature for 2 h, followed by 3 × wash with Dulbecco's PBS. Attached Candida cells were fixed, stained, and counted. Aggregates of Candida cells of larger than four cells were not counted. Cells attached per mm2 of surface were determined in triplicate and are presented as the mean ± S.D. Asterisks (*) indicate that the adhesion induced by hemoglobin significantly differs from the respective control with p < 0.05 by a two-tailed t-test. LN, laminin; FG, fibrinogen; and TSP, thrombospondin-1.

Hemoglobin Induces a Shared Receptor for Fibronectin, Laminin, and Fibrinogen-- Binding of fibronectin to C. albicans induced by hemoglobin was previously shown to be saturable and described by a single class of binding sites (17). Similarly, induced binding of fibrinogen and laminin were also saturable (Fig. 3), but cross-competition experiments suggested that these three proteins partially share a common binding site. Fibrinogen was equally active as an inhibitor of iodinated laminin, fibrinogen, or FN binding, and the binding of all three labeled proteins was completely inhibited by excess fibrinogen (Fig. 3A). This result demonstrates that fibrinogen binds to a site recognized by all three proteins. In contrast, laminin was a better inhibitor of iodinated laminin binding than of fibronectin binding, suggesting that more than one class of receptor(s) interacts with these two proteins (Fig. 3B).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Competitive displacement of ECM protein binding to C. albicans. Unlabeled fibrinogen (panel A) or laminin (panel B) were used as inhibitors of 125I-ECM protein binding to C. albicans. C. albicans (2 × 106), prepared from a 48-h culture grown in YNB medium supplemented with 1 mg/ml hemoglobin, were incubated with radiolabeled ligand and the indicated concentrations of unlabeled proteins at room temperature for 3 h with shaking. The cell suspensions were transferred to microfuge tubes, and the blastoconidia were separated from unbound radioactive ligand by centrifugation through 100 µl of oil. Radioactive fibrinogen (), fibronectin (), or laminin () bound to the cell pellet was quantified in a gamma counter, and binding is presented as the percent of that measured in the absence of inhibitors, mean ± S.D., n = 3.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Competitive displacement of radiolabeled fibrinogen by proteins. 125I-Fibrinogen binding to C. albicans was determined as described in Fig. 3. Unlabeled fibrinogen (), Type I collagen (), laminin (), fibronectin (), or BSA () were added as competitors at the indicated concentrations. 125I-Fibrinogen binding, expressed as a percentage of that in the absence of inhibitors, is presented as mean ± S.D., n = 3.

Expression of Novel Surface Proteins Induced by Hemoglobin-- Since hemoglobin dramatically induced binding of several ECM proteins involving both distinct receptors and a class of promiscuous receptor, we expected that hemoglobin may induce increased or de novo expression of proteins on the surface of Candida cells. To detect these changes in cell surface protein expression, Candida cells grown in medium with or without hemoglobin supplement were labeled with biotin. Labeled surface components were extracted with either dithiothreitol alone or together with lyticase. Equal amounts of protein for each extract were loaded onto SDS gels and detected using peroxidase-streptavidin (Fig. 5). Biotinylated cell surface proteins ranging from 14 to >100 kDa were observed in hemoglobin-induced and noninduced C. albicans. The proteins detected by streptavidin binding should represent exclusively cell wall or cell membrane proteins, since the sulfonated biotin derivative used is not membrane-permeable. This was verified by comparing electrophoretic profiles of surface-labeled cells to those of cells broken using glass beads, which showed few proteins with similar molecular weights (results not shown). Furthermore, the plasma membrane of the cells remained intact throughout the labeling and lyticase/dithiothreitol extraction. Viability of the cells decreased only 1.8% following labeling and extraction of the surface proteins, based on trypan blue exclusion.

View larger version (101K): [in this window] [in a new window]   Fig. 5.   Expression of cell surface proteins in C. albicans induced by hemoglobin. Candida cells grown in the presence (lanes b and d) or absence (lanes a and c) of hemoglobin were harvested, washed, and labeled with biotin. Cell wall components were extracted by treatment with a combination of lyticase and dithiothreitol (lanes a and b) or dithiothreitol alone (lanes c and d). Cell extracts were separated by 10% SDS gel electrophoresis with 8 M urea, transferred to nitrocellulose membrane, blocked with 3% BSA, incubated with streptavidin conjugated with horseradish peroxidase, and visualized using chemiluminescence. Arrows indicate proteins of either with increased amount or de novo expression under the induction of hemoglobin.

Identification of a FN-binding Protein from the Cell Surface-- Several approaches were used to identify which of these induced surface proteins bind to the ECM proteins. Ligand blots of lyticase extracts with ¹²⁵I-fibronectin or fibrinogen identified a 70-kDa protein, but this protein was identified as an impurity in the lyticase.² In preliminary experiments, we observed that modification of exposed amino groups on C. albicans inhibited binding of ¹²⁵I-fibronectin (Fig. 6). This implied that amino groups on a surface protein are required for fibronectin binding to its receptor. We therefore used ligand protection from chemical modification to identify a fibronectin-binding protein. C. albicans cells were incubated with 0, 10, or 100 µg/ml FN for 2 h to allow FN to bind to its receptors on the cell surface. Excess FN was removed, and exposed amino groups on surface proteins were modified using sulfo-SHPP in the presence of the bound FN. After removal of unreacted sulfo-SHPP and dissociation of the bound FN by thorough washing, the cells were biotinylated. By this approach, biotinylation was limited to those amino groups that were protected by the bound FN. Surface proteins were released by dithiothreitol/lyticase digestion, and biotin-labeled proteins were identified by blotting with streptavidin-peroxidase after separation by SDS gel electrophoresis (Fig. 7). Protection by 10 or 100 µg/ml FN resulted in prominent labeling of a 55-kDa protein (Fig. 7, lanes b and c) that was absent in the cells without FN protection (Fig. 7, lane a). At the higher FN concentration, additional bands were revealed at molecular masses of 45 and 66 kDa (Fig. 7, lane c). In contrast to specific protection of the 55-kDa protein, biotin labeling of the 30- and 70-kDa hemoglobin-induced proteins identified in lane d of Fig. 5 were not altered by preincubation with FN (Fig. 7, lanes a-c).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Inhibition of a fibronectin binding activity by biotinylation. Fibronectin binding activity was determined from cells with (modified, striped bar) or without (control, solid bar) biotinylation. FN binding activity from these respective cells was assayed by incubating 2 × 106 Candida cells with 125I-FN in DPBS in a total volume of 200 µl for 3 h at room temperature. The bound radioactivity was quantified as described under "Experiential Procedures" and are presented as mean ± S.D., n = 3.

View larger version (126K): [in this window] [in a new window]   Fig. 7.   Fibronectin protects the fibronectin-binding proteins from propionation of lysine residues. C. albicans cells of late exponential phase were preincubated with either 0 (lane a), 10 (lane b), or 100 (lane c) µg/ml FN for 2 h, and exposed amino groups were blocked by reacting with sulfo-SHPP at the concentration of 0.5 mg/ml in 50 mM sodium bicarbonate, pH 7.8, for 1 h with shaking. The bound FN was removed by thorough washing, and lysyl residues of surface proteins that were masked by FN binding were labeled by biotinylation. Cell surface proteins were then extracted and separated, and biotinylated proteins were visualized as described under "Experimental Procedures." The arrow indicates the 55-kDa fibronectin-binding protein.

View larger version (89K): [in this window] [in a new window]   Fig. 8.   Purification of a fibronectin-binding protein by affinity chromatography. Pre-biotinylated C. albicans cell surface extracts were incubated with Reacti-Gel conjugated with fibronectin. The resultant column was washed, and eluted fractions were dialyzed, freeze-dried, and analyzed by SDS-polyacrylamide gel electrophoresis. Cell surface proteins were visualized by the ECL chemiluminescent detection kit. C. albicans cells grown in hemoglobin-containing defined medium were protected (lane a) or not protected (lane b) by FN at a final concentration of 100 µg/ml as described in Fig. 7. Lane c, unbound surface proteins eluted from FN affinity column; lane d, elution by 0.2 M sodium acetate, pH 4.0; the electro-eluted 55-kDa protein was revealed by silver staining (lane e) or by ECL chemiluminescence (lane f).

	DISCUSSION

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Both solution phase cell binding assays and adhesion to immobilized extracellular matrix proteins demonstrate that growth of C. albicans in a defined medium containing hemoglobin coordinately up-regulates interactions with laminin, fibronectin, fibrinogen, and type IV collagen. This up-regulation is specific in that interactions with at least two other extracellular matrix proteins, type I collagen and thrombospondin-1 are unaffected. Both binding to the proteins in solution and adhesion to the immobilized proteins are increased by growth in the presence of hemoglobin. A larger increase was observed in binding to the soluble proteins than in adhesion to immobilized proteins. This was noted previously using fibronectin (17) and probably reflects a greater sensibility of soluble ligand binding assays to changes in receptor number compared with the multivalent avidity measured in adhesion assays. Growth in the presence of hemoglobin induced increased expression of several proteins on the surface of C. albicans. FN binding to C. albicans protected amino groups on a 55-kDa protein from chemical modification, identifying this surface protein as a candidate for the hemoglobin-induced ECM receptor. A 55-kDa protein was also identified as a potential FN receptor by purification using affinity chromatography on immobilized FN.

To address whether a single promiscuous receptor or a family of receptors with varying specificities are responsible for the enhancement of ECM protein binding by hemoglobin, we performed quantitative analysis of heterologous displacement experiments. By comparing the K_i values for a single protein to inhibit binding of the three labeled proteins, fibronectin, laminin, and fibrinogen, we demonstrated that fibronectin and fibrinogen bind to a common class of sites that overlap only partially with those sites recognizing laminin. Therefore, growth in the presence of hemoglobin induces expression on C. albicans of a class of promiscuous receptors that bind fibronectin, fibrinogen, laminin, and type IV collagen and specific receptors for some ECM proteins, such as laminin. Binding of both fibronectin and fibrinogen to the promiscuous site produces a linear Scatchard plot, indicating that a homogeneous class of receptors accounts for binding of each of these ligands. Because some of these ECM proteins bind to each other, we could not examine heterologous displacement between all pairs of proteins. Using fibrinogen as a tracer for binding to the promiscuous receptor, however, we can show that native type I collagen does not bind to this receptor. Although C. albicans apparently has receptors that mediate adhesion to type I collagen and thrombospondin-1, these receptors are probably distinct from the promiscuous receptor because their binding and ability to promote adhesion are not affected by growth with hemoglobin.

Studies in other organisms provide precedent for both specific and shared promiscuous receptors for ECM proteins. Promiscuous integrins have been identified in mammalian cells. The best defined of these are the platelet integrin IIbIIIa (IIb/3), which binds to fibrinogen, thrombospondin, fibronectin, and von Willebrand factor (25), and the leukocyte 2 integrin CR3 (CD11b/CD18, reviewed in Ref. 26). Mammalian cells also express several families of scavenger receptors that bind multiple ligands (27). Among these, the low density lipoprotein receptor-related protein and CD36 have been demonstrated to bind to several ECM proteins (28-30). Microbial interactions with multiple ECM proteins are also frequently observed. Staphylococcus aureus interacts with fibronectin, laminin, collagens, thrombospondin-1, and elastin (31) reviewed in (32, 33). Some of these interactions are mediated by distinct receptors, but a S. aureus protein that binds several ECM proteins has also been reported (34). Blood stages of the protozoan pathogen responsible for malaria, Plasmodium falciparum, recognize the host proteins thrombospondin-1, CD36, VCAM1, E-selectin, and ICAM1 via a family of related cell surface receptors (35). The promiscuous receptor on C. albicans resembles a mammalian cell scavenger receptor (27) in that the proteins bound are apparently unrelated in sequence, but only a subset of proteins are recognized by the receptor.

Previous studies of extracellular matrix interactions with C. albicans have identified candidate receptors for fibronectin (5, 8), laminin (16), fibrinogen (36), and entactin (37) and an analog of mammalian integrin  subunits that may mediate adhesion to epithelial cells (reviewed in Ref. 15). Limited evidence has been obtained for partial competition between laminin, fibronectin, and entactin for binding to cell wall extracts of C. albicans (37). However, no competition was observed between fibrinogen and the complement receptor (36). Heparin inhibited binding of C. albicans to several ECM proteins, including fibronectin, laminin, and types I and IV collagen. This inhibition does not reflect binding of the glycosaminoglycans to a C. albicans binding site shared with these proteins but probably resulted from sequestration of ligands after binding of heparin to the proteins (38).

Up-regulated expression of potential receptors for selected ECM proteins was observed at the functional level by increased binding activity and adhesion to the immobilized proteins as well as at the protein level as increased expression of several surface proteins on C. albicans, including a 55-kDa protein. Binding of FN to the cells differentially protected the protein from chemical modification (Fig. 7), suggesting that it may directly mediate hemoglobin-induced interactions with fibronectin. The same 55-kDa protein was also identified as a major protein bound to a FN affinity column and eluted with acetic acid. Proteins of 68-72 kDa were identified previously as putative receptors for laminin, fibrinogen, and C3d (9) and are reviewed in (3). Proteins with molecular masses of 60 and 105 kDa were identified as potential FN receptors in uninduced C. albicans by affinity chromatography on immobilized FN (5, 14). Because lyticase was used to release the surface proteins, protease contamination in the lyticase could result in some degradation of the receptor. Thus, the native molecular mass of the receptor identified here may be larger than 55 kDa.

The finding that hemoglobin is a potent regulator of the binding of C. albicans to several ECM proteins may contribute to understanding the pathogenesis of systemic candidasis. Hemoglobin is an abundant circulating protein in the body, and it is often present in sites of tissue injury. C. albicans may readily encounter hemoglobin at sites of tissue injury, which may in turn change its binding to ECM proteins in the tissue. Furthermore, virulent strains of C. albicans express a hemolytic activity that could release hemoglobin from erythrocytes exposed to C. albicans (39). Recently, hyphal cells of C. albicans were reported to bind to human hemoglobin, suggesting that this organism expresses hemoglobin receptors (40). Induction of ECM binding to C. albicans by hemoglobin would facilitate colonization of this organism after it enters the vascular compartment. Therefore, defining of the mechanism by which hemoglobin regulates adherence in this pathogen could identify new targets to prevent or treat C. albicans infections.