Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans

Abstract

A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library ofC. albicanswas screened with serum from mice immunized with ScMp65 (ScW10), aSaccharomyces cerevisiaerecombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract ofC. albicans. After cloning and sequencing of the relevantC. albicanscDNA, 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 (designatedCaMP65) was localized to chromosome R ofC. albicansas 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 inEscherichia coliunder 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 aC. albicansmannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinantC. albicansMp65 but not to ScMp65. Thus, the recombinant Mp65 ofC. albicansretains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.