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Infection and Immunity, December 2000, p. 6777-6784, Vol. 68, No. 12
Department of Bacteriology and Medical Mycology,
Istituto Superiore di Sanità, Rome, Italy
Received 10 July 2000/Returned for modification 3 August
2000/Accepted 31 August 2000
A 65-kDa mannoprotein (CaMp65) has long been studied as a major,
immunodominant antigen of the human opportunistic pathogen Candida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65
(ScW10), a Saccharomyces cerevisiae recombinant protein of
about 48 kDa. This serum recognized the CaMp65 from a cell wall extract
of C. albicans. After cloning and sequencing of the
relevant C. albicans cDNA, an open reading frame encoding a
protein of 379 amino acids was identified. Its deduced amino acid
sequence showed regions of identity with all previously characterized
tryptic fragments of CaMp65, as well as with the corresponding regions
of ScMp65. A prepeptide of 32 amino acids with signal peptidase and
Kex2 cleavage sites as well as a high number of potential
O-glycosylation sites but no N-glycosylation sites or GPI anchor were
observed in sequence studies of CaMp65. A putative adhesin RGD
sequence was also present in the C-terminal region of the molecule.
This triplet was absent in the ScMp65. The relevant gene (designated
CaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat
inducible and associated with germ-tube formation by the fungus. A
recombinant, His6-tagged protein (rCaMp65) was expressed in
Escherichia coli under an inducible promoter. After
purification by nickel-chelate affinity chromatography, the recombinant
product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC)
from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to
induce lymphoproliferation of the PBMC from all the donors. In
addition, a number of CD4+ T-cell clones were generated
using a C. albicans mannoprotein fraction as an antigenic
stimulant. Several of these clones specifically responded to both the
native and the recombinant C. albicans Mp65 but not to
ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for
serodiagnostic and immunological studies.
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Generation of a Recombinant 65-Kilodalton Mannoprotein, a
Major Antigen Target of Cell-Mediated Immune Response to
Candida albicans
*
Corresponding author. Mailing address: Department of
Bacteriology and Medical Mycology, Istituto Superiore di Sanità,
Viale Regina Elena, 299, 00161 Rome, Italy. Phone: 39-06-49387113. Fax: 39-06-49387112. E-mail: cassone{at}iss.it.
Infection and Immunity, December 2000, p. 6777-6784, Vol. 68, No. 12
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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