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.

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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.

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