A seroreactive 120-kilodalton β-1,3-glucanase of Coccidioides immitis which may participate in spherule morphogenesis

Abstract

A β-glucosidase of Coccidioides immitis was identified in electrophoresis gel separations of the concanavalin A-bound mycelial culture-filtrate-plus- lysate preparation. p-Nitrophenol-β-D-glucopyranoside was used as the substrate to visualize the enzymatically active fraction in nonreducing gels. The gel-isolated, chromatographically purified enzyme has an optimal pH of 8.0 and cleaves β-1,3-glycosyl linkages. The alkaline β-glucosidase was further characterized by a pI of 3.8 to 4.0, optimal activity at 37 to 40°C, and molecular size of 120 kDa as identified by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The purified β-glucosidase is identical to a previously reported 120-kDa antigen (Ag) which reacts with immunoglobulin M (IgM) tube precipitin (TP) antibody in sera from patients with coccidioidomycosis. The TP-Ag was described as a valuable serodiagnostic reagent for detection of specific IgM in patients with early coccidioidal infections. The β-glucosidase, like the TP-Ag, was localized in the cell wall and cytoplasmic vesicles of parasitic cells (spherules) by immunofluorescence and immunoelectron microscopy with specific antiserum raised against the purified enzyme. The boiled cell wall fraction isolated from these same young (presegmented) spherules was partially digested by the β-glucosidase. Addition of a potent β-glucosidase inhibitor, 1- deoxynojirimycin, to the parasitic-phase culture medium at a concentration of 200 μM blocked or retarded conversion of arthroconidia to spherules. Antibody was raised in guinea pigs against chromatographically purified 1- deoxynojirimycin which was conjugated with bovine serum albumin. The inhibitor was localized by immunofluorescence in the wall of the 1- deoxynojirimycin-treated cells. We suggest that the spherule wall-associated, alkaline hydrolase functions as a β-1,3-glucanase to provide for wall plasticity as well as intussusception of newly synthesized wall polymers during the period of rapid diametric growth of parasitic cells of C. immitis.