MOLECULAR AND BIOCHEMICAL-CHARACTERIZATION OF A COCCIDIOIDES IMMITIS-SPECIFIC ANTIGEN

Results of earlier investigations have indicated that the saprobic pha se of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific fo r this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and co nfirmed that the secreted proteinase is a Coccidioides-specific antige n (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene,vas localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Ex pression of the csa gene in Escherichia cell using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23- residue signal peptide. The native CS-Ag show ed a low degree of glyco sylation. Analysis of the carbohydrate composition of the CS-Ag reveal ed xylose, mannose, galactose, and glucose. However, the purified anti gen showed no affinity for concanavalin A. A PCR method with specifici ty and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences,were base d on that of the csa gene, was developed. A 520-bp product was amplifi ed only when C. immitis genomic DNA was used as the template. The lowe r limits of DNA detection using this PCR method were 1 pg of C. immiti s genomic DNA by ethidium bromide staining and 100 fg after Southern h ybridization. The csa gene-based PCR method for detection of C. immiti s DNA is useful for culture identification and may have clinical appli cations for the diagnosis of coccidioidal infections.