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Eukaryotic Cell, August 2007, p. 1400-1410, Vol. 6, No. 8
1535-9778/07/$08.00+0     doi:10.1128/EC.00122-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Self-Aggregation of Cryptococcus neoformans Capsular Glucuronoxylomannan Is Dependent on Divalent Cations{triangledown} ,{ddagger}

Leonardo Nimrichter,1,§ Susana Frases,2,§ Leonardo P. Cinelli,3 Nathan B. Viana,4,5 Antonio Nakouzi,2 Luiz R. Travassos,6 Arturo Casadevall,2,7,{dagger}* and Marcio L. Rodrigues1,{dagger}*

Laboratório de Estudos Integrados em Bioquímica Microbiana, Instituto de Microbiologia Professor Paulo de Góes,1 Instituto de Bioquímica Médica,3 LPO-COPEA, Instituto de Ciências Biomédicas,4 Instituto de Física, Universidade Federal do Rio de Janeiro, 21941-590, Brazil,5 Department of Microbiology and Immunology,2 Division of Infectious Diseases of the Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461,7 Disciplina de Biologia Celular, Universidade Federal de São Paulo, São Paulo, SP 04023-062, Brazil6

Received 13 April 2007/ Accepted 1 June 2007

The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca2+ in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.

* Corresponding author. Mailing address for Arturo Casadevall: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2215. Fax: (718) 430-8968. E-mail: casadeva{at}aecom.yu.edu. Mailing address for Marcio L. Rodrigues: Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Suade (CCS), bloco I, 21941590 Rio de Janeiro, Brazil. Phone: 55 21 2562 6740. Fax: 55 21 2560 8344. E-mail: marcio{at}micro.ufrj.br

{triangledown} Published ahead of print on 15 June 2007.

{ddagger} Supplemental material for this article may be found at http://ec.asm.org/.

§ L.N. and S.F. contributed equally to this work.

{dagger} A.C. and M.L.R. share senior authorship of this work.

Eukaryotic Cell, August 2007, p. 1400-1410, Vol. 6, No. 8
1535-9778/07/$08.00+0     doi:10.1128/EC.00122-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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