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Infection and Immunity, December 2000, p. 7049-7060, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Human Antibodies against a Purified Glucosylceramide from Cryptococcus neoformans Inhibit Cell Budding and Fungal Growth

Marcio L. Rodrigues,1 Luiz R. Travassos,2 Kildare R. Miranda,3 Anderson J. Franzen,3 Sonia Rozental,3 Wanderley de Souza,3 Celuta S. Alviano,1,* and Eliana Barreto-Bergter1

Instituto de Microbiologia Professor Paulo de Góes1 and Instituto de Biofísica Carlos Chagas Filho,3 Universidade Federal do Rio de Janeiro, Rio de Janeiro, and Disciplina de Biologia Celular, Universidade Federal de São Paulo, São Paulo,2 Brazil

Received 29 June 2000/Returned for modification 19 July 2000/Accepted 3 August 2000

A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a beta -glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the beta -glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal beta -glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

* Corresponding author. Mailing address: Departamento de Microbiologia Geral, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS-Cidade Universitária, Rio de Janeiro, 21941-590, Brazil. Phone: 55-21-590-3093. Fax: 55-21-560-8344. E-mail: immgceu{at}microbio.ufrj.br.

Infection and Immunity, December 2000, p. 7049-7060, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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