Antigen complex of Coccidioides immitis which elicits a precipitin antibody response in patients

Cole, Garry T.; Kruse, D; Seshan, K. R.

doi:10.1128/iai.59.7.2434-2446.1991

Cited by 29 publications

(31 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…CNBr digestion and amino acid sequencing. Attempts to sequence the purified proteinase failed, even when the initial acetone extraction of the SCWF was omitted (7). We concluded that the proteinase is N terminally blocked.…”

Section: Methodsmentioning

confidence: 94%

Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis

et al. 1992

Self Cite

View full text Add to dashboard Cite

A chymotrypsinlike serine proteinase of Coccidioides immitis with an estimated molecular size of 34 kDa has been shown by immunoelectron microscopy to be associated with the walls of the parasitic cells of this human respiratory pathogen. The proteinase has been suggested to play a role in spherule development. We report the isolation of a 1.2-kb cDNA from an expression library of C. immitis constructed in the XZAP II phage vector. The cDNA is suggested to encode the 34-kDa protein. We demonstrate identity between segments of the deduced amino acid sequence of the open reading frame of the 1.2-kb cDNA and three distinct sequences obtained from cyanogen bromide cleavage peptides of the purified proteinase. The occurrence of N-glycosyl linkage sites in the deduced sequence of 309 amino acids of the open reading frame (ORF) correlates with our identification of such linkage sites in the native glycosylated proteinase. A protein encoded by an 800-bp fragment of the 1.2-kb cDNA, which was produced by transformed Escherichia coli XL1-Blue, was recognized by the anti-34-kDa protein antibody in a Western blot (immunoblot). Northern (RNA) hybridization of total poly(A)-containing RNA of C. immitis with the labeled 1.2-kb cDNA clone revealed a single band of approximately 1.75 kb. Partial homology was demonstrated between the deduced amino acid sequence of the ORF (927 bp) and reported sequences of a-chymotrypsin and chymotrypsinogens. Expression of the proteinase gene was examined by Northern dot blot analysis of total RNA from different stages of parasitic cell development in C. immitis. Maximum levels of specific mRNA were detected during early endospore wall differentiation. The 34-kDa proteinase appears to be concentrated in walls of the parasitic cells at stages of active growth. We suggest that the enzyme may participate in wall plasticization and/or intussusception or in cell wall turnover.

show abstract

Section: Methodsmentioning

confidence: 94%

Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis

et al. 1992

Self Cite

View full text Add to dashboard Cite

show abstract

“…The immunolocalization of the TP antigen has been evaluated in a previous study by Cole et al (4) using human anti-TP antibodies. The antibodies were isolated from patients' sera by immunoadsorption on 3-O-methylmannose, a carbohydrate which has been shown to react with anti-TP antibodies (4,5). In immunoelectron microscopic analyses of presegmented spherules, the investigators reported that the TP antigen was present within the inner cell wall, being detected at the highest concentration along the plasmalemma (4).…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

et al. 1992

View full text Add to dashboard Cite

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...

show abstract

“…The eluate was concentrated by ultrafiltration (Amicon Corp., Danvers, Mass.) as described in an earlier report (8). The concentrated antibody preparation was used for the Western blot analysis described above and for immunoelectron microscopy.…”

mentioning

confidence: 99%

“…Conidia were produced in culture, as previously described (7), and spherules were produced in the lungs of C. immitis-infected BALB/c mice, as reported by Sun et al (36). Conidia and infected lung tissue were chemically fixed, embedded in low-viscosity resin which was cured at 37°C, and thin sectioned in preparation for transmission electron microscopy, as described previously (8). Thin sections were reacted with the isolated anti-124-kDa-FP rabbit antibody diluted 1:2 to 1:10 in blocking buffer and then with goat anti-rabbit IgG conjugated with colloidal gold (15-nm-diameter particles; Sigma), as previously described (9).…”

mentioning

confidence: 99%

Coccidioides immitis fractions which are antigenic for immune T lymphocytes

et al. 1991

Self Cite

View full text Add to dashboard Cite

The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specffic murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay.Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gtll to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-celi response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.

show abstract

Antigen complex of Coccidioides immitis which elicits a precipitin antibody response in patients

Cited by 29 publications

References 34 publications

Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis

Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis

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

Coccidioides immitis fractions which are antigenic for immune T lymphocytes

Contact Info

Product

Resources

About