Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

Abstract

The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris-HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal D-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68kD and 180kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for beta-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues.

Fig. 1

The interaction between macrophage-membrane proteins and histoplasmin components, by ELISA. (a) ELISA of macrophage-membrane proteins interacting with histoplasmin, that reacted with anti-Histoplasma mouse serum (•) or its IgG (▪); ELISA of macrophage-membrane proteins adsorbed with live H. capsulatum yeast cells using histoplasmin/anti-Histoplasma mouse serum to detect specific reactions (○). (b) ELISA of macrophage-membrane proteins enriched by Abrus precatorius (▪) or concanavalin A (▴) as well as macrophage-membrane proteins enriched by A. precatorius adsorbed with live H. capsulatum yeast cells using histoplasmin/anti-Histoplasma mouse serum to detect specific reactions (□). All ELISA readings were corrected with optical density (OD) values for normal serum or IgG.

Fig. 2

The interaction between macrophage-membrane proteins and histoplasmin components, by immunoblotting. Solubilized macrophage-membrane proteins were electrophoresed in SDS–PAGE and electrotransferred to nitrocellulose. After histoplasmin incubation, anti-Histoplasma human serum was added, followed by biotinylated goat anti-human IgG/streptavidin–peroxidase system to reveal the reaction (see details in Materials and Methods). (a) Transference with histoplasmin incubation. (b) Transference without histoplasmin incubation (negative control). I, Immunized mice; N, non-immunized mice. Numbers on left refer to molecular size ( kD).

Fig. 3

Interaction between yeast-adsorbed macrophage-membrane proteins and histoplasmin components, by immunoblotting. Solubilized macrophage-membrane proteins were electrophoresed in SDS–PAGE and electrotransferred to nitrocellulose. After histoplasmin incubation, anti-Histoplasma mouse serum was added, followed by biotinylated goat anti-mouse IgG/streptavidin–peroxidase system to reveal the reaction (see details in Materials and Methods). Lane 1, transference of live H. capsulatum yeast-adsorbed macrophage-membrane protein samples; lane 2, transference of total macrophage-membrane protein samples (positive control). Numbers on left refer to molecular size ( kD).