Isolation of antigens with proteolytic activity from Coccidioides immitis

Abstract

Three antigens with proteolytic activity have been isolated from crude, water-soluble fractions of the saprobic phase of the fungal pathogen Coccidioides immitis. Two proteinases, identified in our immunoelectrophoresis reference system as Agll and AgCS, were isolated from the soluble conidial wall fraction (SCWF). Agll was previously shown to be a serine proteinase and was characterized in this study as a 60-kilodalton (kDa) fraction by gel filtration (GF). The purified proteinase demonstrated little or no reactivity with 21 serum samples from coccidioidomycosis patients in the enzyme-linked immunosorbent assay; this may be due to limited presentation of this antigen to the host during the course of coccidioidomycosis. AgCS was separated by GF chromatography into two fractions identified by molecular masses of 39 and 19 kDa. Most proteolytic activity was shown by substrate gel electrophoresis to be associated with the lower-molecular-mass fraction. AgCS was reactive with 18 of the 21 serum samples and shown to be the major component of a heat-stable antigen previously reported to be immunospecific for C. immitis. The third antigen with proteolytic activity was isolated from the 5-day mycelial culture filtrate and identified by GF as a 56-kDa fraction. Uniformly high levels of immunoreactivity between 18 of the 21 patient sera and the 56-kDa antigen were demonstrated. Antigens with proteolytic activity may play important roles in fungus-host interactions as well as morphogenesis of the pathogen.

The coccidioidal complement fixation and immunodiffusion-complement fixation antigen is a chitinase

“…Recently, the M antigen of Histoplasma capsulatum was identified as a catalase (8). Cole et al (5) isolated three proteolytic antigens (unknown serodiagnostic activity) from the mycelial phase of C. immitis, and Resnick et al (25,26) isolated a 21-kDa protein which had proteolytic activity and tentatively appeared to correspond to the IDTP antigen. Indeed, there are other examples, including the enolase of Candida albicans, which is the 48-kDa immunodominant protein (20,30).…”

Virulence factors of medically important fungi

“…Cole et al identified two additional proteinases by substrate gel electrophoresis of crude antigen preparations (49). One proteinase isolated from 5-day-old mycelial culture filtrates cannot degrade immunoglobulins and has a molecular mass of 56 kDa estimated by gel filtration.…”

Coccidioides immitis fractions which are antigenic for immune T lymphocytes

“…The materials with higher and lower molecular iiasses were distinctly less stimulatory. This suggests that previously characterized proteins which elicit at least some antibody response and have been isolated from the SCWF, such as a chymotrypsin-like serine proteinase (13) and 19-kDa proteinase (5,13), do not stimulate proliferation of these T cells.…”

“…The concentrated antibody preparation with affinity for the T-cell-reactive FP was diluted 1:5 in blocking buffer. In each case, bound antibody was detected by incubation of the NC membranes with goat anti-rabbit IgG (heavy + light chains) conjugated to peroxidase (Kirkegaard & Perry), as previously described (13). Control immunoblots included incubations of anti-SCWF with the SDS-PAGE separation of the control lysate and incubation of the anti-FP antibody with electrophoretic separations of the recombinant lysate which contained the homologous FP (see Fig.…”

“…The identified T-cellreactive FP (124 kDa) (see Fig. 4) was isolated by electroelution from SDS-polyacrylamide gels after electrophoretic separation of the lysate, as previously described (13). The isolated 124-kDa FP was then coupled to cyanogen bromide (CNBr)-activated Sepharose 4B (Pharmacia), and the subsequent washing, serum binding, and elution steps were the same as previously described (7).…”