Characterization of 3- O -Methylmannose from Coccidioides immitis

Abstract

3-O-methylmannose isolated from Coccidioides immitis was characterized by gas chromatographic comparison of derivatives with those of synthetic 3-O-methylmannose. Gas chromatographic behavior of three derivatives of the natural and synthetic sugars was identical.3-O-methylmannose has been identified in Streptomyces griseus (2), Escherichia coli 08 (3), Klebsiella type K 57:05 (3), and Klebsiella types 2 and 6 (Gormus and Wheat, unpublished data) and was tentatively identified in Coccidioides immitis (5). The present work documents characterization of the C. immitis sugar as 3-0-methylmannose.The naturally occurring methylated sugar was obtained from pooled, dialyzed culture filtrates of C. immitis Silveira strain. The fungus was grown in glucose-yeast dialysate medium in a shaking broth culture for 3 days at 37 C. Culture filtrate (spent medium) was separated from the mycelial mat by filtration through Whatman no. 1 paper on a Buchner funnel. Culture filtrate solubles were passed through a Selas candle (porosity 03) to insure sterility, dialyzed against deionized water, and lyophilized. The lyophilized retentate (150 mg) was hydrolyzed with 15 ml of 2 N hydrochloric acid for 2 hr at 100 C, and acid was removed by distillation under reduced pressure. The hydrolysate was deionized with Dowex 50-H+ and then with Dowex 1-HC03-, concentrated, and then streaked on six thin-layer plates coated with a 0.5-mm layer of microcrystalline cellulose (Machery, Nagel & Co.). Glucose and synthetic (1) 3-0-methyl-Dmannose were run on either side of the hydrolysate streak. The plates were developed twice in n-butanol-acetic acid-water (5:1:2) and then sprayed with alkaline silver nitrate or Timell's reagent as described previously (5) to locate the standard sugars and the edges of the hydrolysate bands. Bands with RGIC values corresponding to synthetic 3-0-methylmannose were scraped from the plates and eluted with water. The slurry was filtered on Whatman no. 1 paper, and the filtrate was passed through a 0.45-Am filter disc (Milli-pore Corp.) before being lyophilized (yield, 4.1 mg, 2.7%).In an earlier report (5), tentative identification of the natural methylated sugar was made by thin-layer chromatography. Gas chromatography has now been used to compare several derivatives of the natural and synthetic sugars. The major peaks observed on gas-liquid chromatograms of the natural and synthetic preparations were found to be additive in all cases. The derivatives were prepared by (i) conversion of the sugars to their alditol acetates, (ii) preparation and acetylation of the methyl glycosides, and (iii) acetylation of the sugars without prior reduction.The alditol acetates were prepared by the method of Sawardeker et al. (4). For preparation of the acetylated methylglycosides, a 20-,ug sample of the sugar was heated under reflux for 2 hr with 1 ml of 2% methanolic hydrogen chloride. The solvent was removed in a rotary evaporator, and the residue was then heated at 138 C with 0.25 ml of an acetic anhydride-pyridine mixture (2:1, v/v) fo...

Comparison of Coccidioides immitis arthrospore, mycelium, and spherule cell walls, and influence of growth medium on mycelial cell wall composition

“…1). The same sugars were previously reported in mycelial culture filtrates (e.g., references 2, [16][17][18]. Approximate relative quantities of neutral sugars in the cell wall fractions were determined as alditol acetates by GLC ( Table 6).…”

“…(v) 3-0-methylmannose. This compound was identified by thin-layer cellulose chromatography and by GLC of the alditol acetate, using chemically synthesized reference compound as previously reported (18).…”

“…Most analyses reported pertain to the carbohydrate and nitrogen content of coccidioidin preparations, whether from mycelia, mycelial culture filtrates (i.e., coccidioidin) contain autolysates (13). Mycelial autolysates and culture filtrates (i,e., coccidioidin) contain ethanol-precipitable polymers composed predominantly of mannose, glucose, 3-0-methylmannose, and protein (2, 11,[15][16][17][18], but the presence of galacturonic acid (11,15) was not confirmed (2,16,17). Spherule autolysates of a variety of strains were reported to contain carbohydrate as mannose, protein, and a high content of nonprotein nitrogen (e.g., 13).…”

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).…”

Serology of coccidioidomycosis.

“…1958, P. 98, p. 127) confirmed the presence of mannose and galactose; they, too, found an unknown sugar which they reported as monomethylmannose. Later, this was shown to be 3-O-methylmannose accompanied by glucose as well as mannose and galactose (119,131). Cole et al (21) studied the dialyzed retentate of 16-day-old mycelial culture filtrates by gas-liquid chromatography.…”