Isolation and purification of antigenic components of Cryptococcus

Abstract

The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying the antigenic, biochemical, and virulence properties of Cryptococcus species.

Fig. 7.1

Shown are the different structures for the glucuronoxylomannan (GXM) of Cryptococcus serotypes A to D. Serotypes A and D belong to C. neoformans, and serotypes B and C belong to C. gattii. The mannose backbone of GXM has variable degrees of O-acetylation (not shown). GlcpA, glucopyranosyluronic acid; Manp, mannopyranan; Xylp, xylopyranosyl. (Reproduced and adapted with permission from Cherniak, R., Valafar, H., Morris, L.C., and Valafar, F. 1998. Cryptococcus neoformans Chemotyping by Quantitative Analysis of 1H Nuclear Magnetic Resonance Spectra of Glucuronoxylomannans with a Computer-Simulated Artificial Neural Network. Clin. Diagn. Lab. Immunol. 5, 146–159.)

Fig. 7.2

Mannoprotein Cda1/MP-98 Cryptococcus neoformans is shown as a model mannoprotein. The major defining characteristics of MPs are their S/T rich region and their GPI anchor. The putative N-glycans are indicated by the branching structures, and the putative O-glycans are indicated by the straight dotted structures in the S/T rich regions. (Reproduced and adapted with permission from Levitz, S. M., and Specht, C. A. 2006. The molecular basis for the immunogenicity of Cryptococcus neoformans mannoproteins. FEMS Yeast Research 6, 513-524.)

Fig. 7.3

Diagram of the mannoprotein concentration system. Culture supernatant is placed in the lower flask, pumped through a tangential filtration cassette, and concentrated before MP purification. (Figure courtesy of Dr. Michael Mansour.)

Fig. 7.4

Analysis of C. neoformans strain CAP67 supernatant fractions by SDS-PAGE. Crude (CR), flow-through (FT), and MP fractions were resolved by 12% SDS-PAGE and analyzed by (A) silver stain, (B) PAS, and (C) Con A–biotin blot. Migration of commercial molecular mass standards, expressed in kilodaltons, is indicated to the left of the gels. (Reproduced and adapted with permission from Mansour, M. K., Schlesinger, L.S., and Levitz, S. M. 2002. Optimal T cell responses to Cryptococcus neoformans mannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors. Journal of Immunology 168, 2872–2879.)