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

Abstract

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.

Virulence factors of medically important fungi

“…The gene encoding this serine proteinase has been cloned and sequenced, and its B-cell epitopes were expressed on a fusion protein encoding a portion of the mature enzyme (48). The open reading frame of the gene predicts a protein of 34.3 kDa, which is in agreement with the 36 kDa observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels.…”

“…The association of the proteinase with mycelia and conidia suggests that it may be released from outer cell wall of conidia soon after inhalation, leading to immediate damage of respiratory tract tissue (323). Immunoelectron microscopy with rabbit monospecific antiserum against purified proteinase detected the proteinase in the cell walls of mycelia and spherules, in the segmentation apparatus of the developing spherule (324), and in close association with chitin in the presegmentation spherule (48). The density of the proteinase in the segmentation apparatus suggests that one likely role of the proteinase in the fungal life cycle is to promote the dissolution of the wall matrix and segmentation apparatus as the endospores mature.…”

History of medical mycology in the united states

“…The suggested molecular size of the purified proteinase was 34 kDa. The enzyme was localized in the walls of parasitic cells of C. immitis (immunoelectron microscopic studies) (63). In 1992, Cole et al also reported isolation and expression of the gene that encoded the enzyme (63).…”

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

“…The highest enzyme activity was observed in the presence of presegmented spherule wall material. The first-generation spherules (strain C735) from which this wall fraction was obtained were incubated for approximately 36 h in modified liquid Converse medium and were nearly synchronized in their early diametric growth phase (9). Wall preparations in the absence of ,B-glucosidase showed no glucose release.…”

“…Certain of these isolated cell wall-associated antigens (Ags) also demonstrate proteolytic activity (10). Proteinases expressed by the parasitic phase have been suggested to play roles in spherule development and endosporulation as well as host tissue invasion and destruction (9,32).…”