Hemoglobin-induced binding of Candida albicans to the cell-binding domain of fibronectin is independent of the Arg-Gly-Asp sequence

Abstract

Hemoglobin specifically induces fibronectin (FN) binding to the pathogenic yeast Candida albicans. When grown in the complex medium Sabouraud broth, C. albicans expresses receptors that bind to several domains of FN. Growth in defined medium supplemented with 0.1% hemoglobin, however, enhanced the binding of FN to a single class of receptors, with a Kd = 4.6 x 10(-8) M. Competitive binding assays using recombinant and proteolytic fragments of FN revealed that the cell-binding domain mediated this interaction. A recombinant 40-kDa fragment of FN consisting of type III repeats 9 to 13 had an inhibitory activity similar to that of the entire 120-kDa cell-binding domain, indicating that the C-terminal portion of the cell-binding domain contains the binding site. A recombinant 33-kDa fragment of the cell-binding domain and a 33-kDa fragment with the RGD sequence deleted had the same inhibitory activities, demonstrating that the RGD sequence recognized by some mammalian integrins is not required. The addition of hemoglobin to the culture medium also enhanced Candida cell adhesion to immobilized FN and to 120- and 40-kDa fragments of FN but not to the collagen-binding or fibrin I domains. Using ligand protection, we identified a surface protein from C. albicans with an apparent molecular mass of 55 kDa that was protected by both FN and the 40-kDa fragment derived from the cell-binding domain. Therefore, hemoglobin both induces FN binding and changes the relative affinities of C. albicans for the cell- and collagen-binding domains of FN.

FIG. 1

FN and proteolytic and recombinant fragments derived from FN. I, II, and III represent, respectively, type I, type II and type III repeating motifs. Proteolytic fragments are indicated by the prefix p, and recombinant fragments are indicated by the prefix r. Numbers following p or r indicate the molecular mass in kilodaltons. The arrows indicate the positions of cell adhesion recognition sequences RGD, LDV, and REDV. The asterisk indicates the position of the RGD sequence deletion from the r33 fragment (r33Δ).

FIG. 2

Competitive displacement of FN and its derived fragments binding to C. albicans. Unlabeled FN (○), the proteolytic 120-kDa cell-binding domain (•), and proteolytic 30- (▿) and 40-kDa fragments (▴) were used as inhibitors of ¹²⁵I-FN binding to C. albicans. C. albicans cells (2 × 10⁶), prepared from a 48-h culture grown in YNB medium supplemented with 1 mg of hemoglobin per ml, were incubated with radiolabeled ligand and the indicated concentrations of unlabeled proteins at 26°C for 3 h. Binding is presented as the percentage of that measured in the absence of inhibitors; values are means ± SD (n = 3).

FIG. 3

Growth of C. albicans in hemoglobin enhances adhesion to the cell-binding domain of FN. Candida cells prepared from cultures with (shaded bars) or without (solid bars) hemoglobin were allowed to adhere to FN or FN fragments applied to Chamber slides as described in Materials and Methods. The numbers of cells attached per square millimeter of surface were determined in triplicate and are presented as the means ± SD. An asterisk indicates that the adhesion induced by hemoglobin significantly differs from that induced by the respective control, with P < 0.05 by a two-tailed t test.

FIG. 4

Dose dependence of cell adhesion to immobilized FN or its fragments. FN (▿) and FN fragments p120 (○), p30 (•), r31 (□), and r40 (▴) were adsorbed at 0.01, 0.1, 1, and 10 μg/ml in DPBS without Ca²⁺ and Mg²⁺ (pH 7.5) to Chamber slides by incubation overnight at 4°C. Unbound protein was removed by washing the wells three times with DPBS. Candida cells prepared from cultures with hemoglobin in YNB medium were added to each well. Attached Candida cells were fixed, stained, and counted (n = 3).

FIG. 5

Identification of FN-binding proteins by ligand protection. C. albicans cells at late-exponential phase were preincubated without ligand (lane b), with the r40 fragment of FN (lane a), or with FN (lane c) at 100 μg/ml for 2 h, and exposed amino groups were blocked by reacting them with sulfo-SHPP at a 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 then labeled by biotinylation. Cell surface proteins were then extracted and separated, and biotinylated proteins were visualized as described in Materials and Methods.