Fractionation and Composition Studies of Skin Test-Active Components of Sensitins from Coccidioides immitis

Abstract

Coccidioidin skin-test activities from mycelial culture filtrates and autolysates were partially purified. Major chemical constituents included 3-O-methylmannose, mannose, and amino acids. Coccidioidin is the filtrate of liquid cultures of Coccidioides immitis which is used as a skin test and serological antigen. Cooke, in 1915 (1), reported equivocal results with several "emulsions" and heat-concentrated glucose broth filtrates. The first successful coccidioidin, prepared by Davis in 1924 (3), was a suspension of the fungus rather than a filtrate. However, Hirsch and D'Andrea, in 1927 (7), reported skin reactions of the tubercuin this study was glucose and yeast extract dialysate (8). After checking for contamination by both culture and microscope examination, mycelia and liquids were separated by filtration (8). The culture filtrate was passed through a Selas Candle, porosity 03 (Selas Corp. of America, Spring House, Pa.), and evaporated under reduced pressure (20 mm or less) and temperature (40 C or less). After concentration, the culture filtrate was dialyzed against deionized water for 24 to 48 hr, lyophilized, weighed, and stored in a desiccator. The mycelium was allowed to autolyze in aqueous 3% toluene for 7 days at 37 C and harvested by Buchner funnel filtration on Whatman no. 1 paper to separate the particulate residue from the toluene-autolyzed soluble materials. The autolysate was then passed through a Selas Candle. The results of the investigation reported in this study are of the two soluble sensitins obtained as the culture filtrate and toluene autolysate. 294

Immunoaffinity isolation and partial characterization of the Coccidioides immitis antigen detected by the tube precipitin and immunodiffusion-tube precipitin tests

“…DEAE peak 1 (unbound) contained the monosaccharides 3-O-methylmannose, mannose, and glucose in a ratio of 8:1.2:1; DEAE peak 2 (bound) contained the monosaccharides 3-O-methylmannose, mannose, glucose, and galactose in a ratio of 1:1:1:1. These four monosaccharides had previously been shown in the culture filtrate, cell walls, and nonwall polymer material of C. immitis (1,21,23,(33)(34)(35). The size range of these two glycopeptides confirmed our earlier gel filtration studies of ID-TP activity (38).…”

Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody

“…C-ASWS-M and C-ASWS-S are both composed of protein and polysaccharide but differ in that C-ASWS-M contains more protein; C-ASWS-S contains a greater amount of polysaccharide, and of the polysaccharide constituents, 3-0-methylmannose is present in an almost fivefold increased concentration in C-ASWS-S. The latter finding is of particular interest because of the association of 3-0- (1,11,24,25,32).…”

Occurrence of antigenic (species-specific?) partially 3-O-methylated heteromannans in cell wall and soluble cellular (nonwall) components of Coccidioides immitis mycelia

“…Organism. C. immitis Rixford and Gilchrist 18%, strain Silveira, was grown at 34°C on 2% glucoseyeast extract dialysate with shaking for 3 to 7 days as previously described (1). No arthroconidia or spherules were observed.…”

“…Reference preparations obtained as solutions were used at the following dilutions: CDN-b was a 1:10 dilution of C. E. Smith's 64 D4 (graciously donated by D. Pappagianis, School of Medicine, University of California, Davis), CDN-c was a 1:10 dilution of commercial mycelial CDN solution (obtained from latrogenics, Tempe, Ariz.), and SPN was a 1:10 dilution of a "High Test Strength" SPN preparation (obtained from Berkeley Biologicals, Berkeley, Calif.). CDN-a was a lyophilized culture filtrate prepared and calibrated for activity by dose-response tests in previous work in this laboratory (1,16,17).…”

“…These preparations are the phenol-soluble, water-soluble (PSWS) extracts of culture filtrates or of whole mycelial cells (16)(17)(18) and the alkalisoluble, water-soluble (ASWS) extracts of trypsinized cell walls of mycelia or spherules (4,15). The PSWS extracts have been shown to contain 3-0-methylmannose, mannose, small amounts of galactose, and variable amounts of glucose, protein, and other undefined components (1,17). A comparable characterization of the composition of the ASWS extracts has not been presented.…”