Localization of the tube precipitin and complement fixation antigens of Coccidioides immitis by immunoelectron microscopy with murine monoclonal antibodies

Abstract

The cellular localization of the tube precipitin (TP) and complement fixation (CF) antigens of Coccidioides immitis was examined by immunoelectron microscopy with murine immunoglobulin Gl monoclonal antibodies directed against the TP and CF antigens, respectively. Immunoelectron microscopic analyses of saprobicand parasitic-phase cells showed that the TP antigen is present at a high concentration within the inner cell wall layer and along the plasma membrane. The antigen was also detected, at a lesser concentration, within cytoplasmic vacuoles. In contrast to the predominant localization of the TP antigen in the cell walls, the CF antigen resides primarily within the cytoplasm, where it appears to be dispersed throughout the cytoplasm rather than associated with a specific cytoplasmic organelle. A sparse amount of the CF antigen within the inner cell walls was also demonstrable. The localization of the TP and CF antigens throughout the morphogenetic phases of C. immitis has important implications in antigen production and in analyses of host response in coccidioidomycosis.Serologic detection of the tube precipitin (TP) and complement fixation (CF) antibodies to Coccidioides immitis provides an extremely valuable aid in establishing the diagnosis and clinical stage of coccidioidomycosis (17)(18)(19). Production of the TP antibody occurs early after primary infection, usually within 1 to 3 weeks of clinical onset, and diminishes to negligible levels by the 4th month of illness. The CF antibody is produced later in the disease, generally within 3 to 4 months, and persists throughout active infection. The magnitude of the CF antibody response, as measured by serial dilutions of the patient's serum or other biofluid, provides an index of disease involvement. Low titers generally reflect localized disease involving the lungs or a single extrapulmonary site, whereas high titers are consistent with extensive multifocal disease.Previous investigations have established that the TP antigen is a high-molecular-weight, heat-stable polysaccharide or polysaccharide-protein complex (4,5,8,9,15,25) which can be extracted from cell walls by enzymatic treatment (7) or by incubating the walls in 1 N NaOH (10). The CF antigen has been characterized as a heat-labile protein or glycoprotein (8,12,23,24), and although its cellular localization has not been established, evidence suggests that it is of cytoplasmic origin (7,17,23,24). In order to more clearly define the cellular localization of the TP and CF antigens, we performed immunoelectron microscopic analyses of the morphogenetic phases of C. immitis by using a murine monoclonal antibody (MAb) which recognizes a heat-stable carbohydrate epitope on the TP antigen (13) and a murine MAb which is directed against a heat-labile peptide epitope on the CF antigen (12). * Corresponding author. MATERIALS AND METHODSFungi. C. immitis Silveira (ATCC 28868) was cultured under previously described conditions for the production of saprobic and parasitic cells (6,22). In brief, mycelia were grown...

Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen

“…The first 17 amino acids on the CF/chitinase cDNA product were lacking on the mature CF/chitinase protein, indicating that this portion of the cDNA was not translated or, alternatively, was cleaved posttranslationally. The latter possibility is supported by the similarity in the composition of the 17-amino-acid sequence with that of signal peptides, namely, a positively charged residue (Arg-2) at the N terminus, a stretch of hydrophobic amino acids, and a putative signal peptidase cleavage site (Val-Glu-Ala) (7,20,28). If there is no additional processing of the CF/chitinase protein after cleavage of the 1.8-kDa signal peptide, the mature CF/chitinase protein would have a size of approximately 45 kDa, which is concordant with our previous report that the secreted antigen is detected as a 43-to 45-kDa couplet band in SDS-polyacrylamide gels and immunoblots (8).…”