Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 Jan;59(1):59–64. doi: 10.1128/iai.59.1.59-64.1991

Facilitated isolation, purification, and analysis of glucuronoxylomannan of Cryptococcus neoformans.

R Cherniak 1, L C Morris 1, B C Anderson 1, S A Meyer 1
PMCID: PMC257705  PMID: 1987064

Abstract

Cryptococcus neoformans was cultured in a chemically defined medium. The culture was adjusted to 0.25% formaldehyde or autoclaved after 5 days of growth at 35 degrees C, and a cell-free supernatant was obtained by centrifugation. Solid calcium acetate was added to the supernatant to give a 5% solution, and the pH was adjusted to approximately 5 with glacial acetic acid. The polysaccharide (PS) was precipitated by the addition of 3 volumes of 95% ethanol. The PS was dissolved in 0.2 M NaCl, and insoluble calcium salts were solubilized by the addition of several drops of glacial acetic acid. The PS solution was treated by ultrasonic irradiation for 15 min. This concurrently decreased the molecular weight of the PS and reduced the viscosity of the solution. The ultrasonically irradiated PS was precipitated by differential complexation with hexadecyltrimethylammonium bromide at 23 degrees C, the complex was dissolved in 1 M NaCl, and the glucuronoxylomannan was precipitated by adding 3 volumes of ethanol. The glucuronoxylomannan was dissolved in 1 M NaCl and then ultrasonically irradiated for 2 h to reduce the molecular mass to a limiting value of approximately 100 kDa (GXMS). The purified GXMS was centrifuged, dialyzed, and finally recovered by lyophilization. GXMS was chromatographed on DEAE-cellulose at reasonable concentrations without the complication of high solution viscosity. The sugar composition and structure of GXMS were determined by gas-liquid chromatography, permethylation gas-liquid chromatography-mass spectrometry, and 13C nuclear magnetic resonance spectroscopy. The improved solution characteristics of GXMS were ideal for the determination of its chemical and serological properties.

Full text

PDF
62

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BENNETT J. E., HASENCLEVER H. F. CRYPTOCOCCUS NEOFORMANS POLYSACCHARIDE: STUDIES OF SEROLOGIC PROPERTIES AND ROLE IN INFECTION. J Immunol. 1965 Jun;94:916–920. [PubMed] [Google Scholar]
  2. Bhattacharjee A. K., Bennett J. E., Glaudemans C. P. Capsular polysaccharides of Cryptococcus neoformans. Rev Infect Dis. 1984 Sep-Oct;6(5):619–624. doi: 10.1093/clinids/6.5.619. [DOI] [PubMed] [Google Scholar]
  3. Blandamer A., Danishefsky I. Investigations on the structure of the capsular polysaccharide from Cryptococcus neoformans type B. Biochim Biophys Acta. 1966 Apr 25;117(2):305–313. doi: 10.1016/0304-4165(66)90081-x. [DOI] [PubMed] [Google Scholar]
  4. Blumenkrantz N., Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem. 1973 Aug;54(2):484–489. doi: 10.1016/0003-2697(73)90377-1. [DOI] [PubMed] [Google Scholar]
  5. Breen J. F., Lee I. C., Vogel F. R., Friedman H. Cryptococcal capsular polysaccharide-induced modulation of murine immune responses. Infect Immun. 1982 Apr;36(1):47–51. doi: 10.1128/iai.36.1.47-51.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bulmer G. S., Sans M. D. Cryptococcus neoformans. 3. Inhibition of phagocytosis. J Bacteriol. 1968 Jan;95(1):5–8. doi: 10.1128/jb.95.1.5-8.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cherniak R., Cheeseman M. M., Reyes G. H., Reiss E., Todaro F. Enhanced binding of capsular polysaccharides of Cryptococcus neoformans to polystyrene microtitration plates for enzyme-linked immunosorbent assay. Diagn Clin Immunol. 1988;5(6):344–348. [PubMed] [Google Scholar]
  8. Cherniak R. Soluble polysaccharides of Cryptococcus neoformans. Curr Top Med Mycol. 1988;2:40–54. doi: 10.1007/978-1-4612-3730-3_2. [DOI] [PubMed] [Google Scholar]
  9. Darvill A. G., McNeil M., Albersheim P. Structure of Plant Cell Walls: VIII. A New Pectic Polysaccharide. Plant Physiol. 1978 Sep;62(3):418–422. doi: 10.1104/pp.62.3.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dismukes W. E. Cryptococcal meningitis in patients with AIDS. J Infect Dis. 1988 Apr;157(4):624–628. doi: 10.1093/infdis/157.4.624. [DOI] [PubMed] [Google Scholar]
  11. HAKOMORI S. A RAPID PERMETHYLATION OF GLYCOLIPID, AND POLYSACCHARIDE CATALYZED BY METHYLSULFINYL CARBANION IN DIMETHYL SULFOXIDE. J Biochem. 1964 Feb;55:205–208. [PubMed] [Google Scholar]
  12. Jacobson E. S., Ayers D. J., Harrell A. C., Nicholas C. C. Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans. J Bacteriol. 1982 Jun;150(3):1292–1296. doi: 10.1128/jb.150.3.1292-1296.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. James P. G., Cherniak R., Jones R. G., Stortz C. A., Reiss E. Cell-wall glucans of Cryptococcus neoformans Cap 67. Carbohydr Res. 1990 Apr 2;198(1):23–38. doi: 10.1016/0008-6215(90)84273-w. [DOI] [PubMed] [Google Scholar]
  14. Kozel T. R., Cazin J. Nonencapsulated Variant of Cryptococcus neoformans I. Virulence Studies and Characterization of Soluble Polysaccharide. Infect Immun. 1971 Feb;3(2):287–294. doi: 10.1128/iai.3.2.287-294.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kozel T. R., Mastroianni R. P. Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. Infect Immun. 1976 Jul;14(1):62–67. doi: 10.1128/iai.14.1.62-67.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mort A. J., Parker S., Kuo M. S. Recovery of methylated saccharides from methylation reaction mixtures using Sep-Pak C18 cartridges. Anal Biochem. 1983 Sep;133(2):380–384. doi: 10.1016/0003-2697(83)90098-2. [DOI] [PubMed] [Google Scholar]
  17. Small J. M., Mitchell T. G., Wheat R. W. Strain variation in composition and molecular size of the capsular polysaccharide of Cryptococcus neoformans serotype A. Infect Immun. 1986 Dec;54(3):735–741. doi: 10.1128/iai.54.3.735-741.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Todaro-Luck F., Reiss E., Cherniak R., Kaufman L. Characterization of Cryptococcus neoformans capsular glucuronoxylomannan polysaccharide with monoclonal antibodies. Infect Immun. 1989 Dec;57(12):3882–3887. doi: 10.1128/iai.57.12.3882-3887.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Turner S. H., Cherniak R., Reiss E. Fractionation and characterization of galactoxylomannan from Cryptococcus neoformans. Carbohydr Res. 1984 Feb 15;125(2):343–349. doi: 10.1016/0008-6215(84)85172-1. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES