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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
-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
-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
-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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