Biochemical and Immunological Characterization of MP65, a Major Mannoprotein Antigen of the Opportunistic Human Pathogen Candida albicans

Abstract

In the search of the antigenic determinants of a 65-kDa mannoprotein (MP65) of Candida albicans, tryptic fragments of immunoaffinity-purified MP65 preparations were tested for their ability to induce lymphoproliferation of human peripheral blood mononuclear cells (PBMC). Five major peptides (T1 to T5) were shown to induce a vigorous proliferation of PBMC from the majority of the eight healthy human subjects tested. With the use of synthetic peptides, critical amino acid sequences of the two most immunoactive (T1 and T2) peptides were determined. Similar to what was found for the MP65 molecule, no PBMC multiplication was induced by the antigenic peptides in cultures of naive cord blood cells. The amino acid sequence analysis of tryptic and chymotryptic peptides of MP65 demonstrated a substantial homology with the deduced sequences of two cell wall proteins of Saccharomyces cerevisiae, encoded by the genes YRM305C and YGR279C. However, the antigenic peptides were those showing the least similarity with the corresponding regions of the above proteins. In particular, the lymphoproliferation-inducing sequence of the T1 peptide scored only 20% identity with the homologous regions of S. cerevisiae proteins. Besides disclosing the amino acid sequence of MP65, this study provides an initial characterization of some of its antigenic determinants, as well as of synthetic peptides of potential use to detect specific immune responses against MP65, a major target of anticandidal cell-mediated immunity in humans.Yeasts belonging to the genus Candida are major fungal pathogens for the immunocompromised host. Candida albicans is especially prevalent in subjects infected by the human immunodeficiency virus with severe functional and numerical deficits in CD4 ϩ T lymphocytes, which are critical components of cell-mediated immunity (CMI) (24). Despite the increased awareness of the important role that CMI plays in the defense against this fungal pathogen (reviewed in references 1 and 38), very few CMI antigen targets for it have so far been identified. These include heat shock proteins, enolase, and a number of as yet uncharacterized mannoproteins, some with adhesin function (6,12,15,27,29,30,40).We have long been studying a 65-kDa mannoprotein (designated MP65) which is present in both the structural and secretory mannoprotein material and which is recognized by T cells of peripheral blood of practically all healthy individuals (quasiuniversal antigen) (20,(42)(43)(44). In mice immunized with whole fungal cells or MP65-rich mannoprotein extract (MP-F2) (44), a vigorous lymphoproliferative response with a prevalent T-helper type 1 (Th1) cytokine pattern was elicited in in vitro MP65-stimulated lymphomonocyte cultures (31). In addition, the MP-F2 extract was capable of inducing a moderate but significant degree of protection against a challenge with a highly virulent C. albicans strain in a model of murine disseminated candidiasis. This protection was significantly increased by coadministration of interleukin-12 (IL-12) or by treatm...

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

“…A strong homology at the protein level was found between this protein and the glucanase or transglycosidase family of cell wall proteins of Saccharomyces cerevisiae (8,20,21). Interestingly, the least similarity between CaMp65 and the yeast proteins was found in the most antigenic peptides of the N-terminus regions of CaMp65 (21).…”

“…The amino acid sequence of the deduced CaMp65 showed regions identical or similar to all previously characterized tryp- tic fragments of the native MP65 protein (21) and, of course, with the corresponding regions of the Scw10 protein (Fig. 2).…”

“…A strong homology at the protein level was found between this protein and the glucanase or transglycosidase family of cell wall proteins of Saccharomyces cerevisiae (8,20,21). Interestingly, the least similarity between CaMp65 and the yeast proteins was found in the most antigenic peptides of the N-terminus regions of CaMp65 (21). The availability of the amino acid sequences of several tryptic and chymotryptic fragments of CaMp65 and the established sequence homology with S. cerevisiae cell wall proteins have allowed us to clone the relevant genes of both C. albicans and S. cerevisiae and to express them in Escherichia coli.…”

Mannoprotein fromCryptococcus neoformansPromotes T-Helper Type 1 Anticandidal Responses in Mice

“…When specifically examined, stimulatory properties of MPs in cell-mediated immunity (CMI) were found to be expressed by the protein rather than by the saccharide moiety of these molecules (7,8,18).…”

“…MPs are abundant in the cell wall of C. albicans and constitute about one-third of the dry mass (3). One of them, an immunodominant 65-kDa-MP antigen (MP65), has been extensively characterized both biochemically and immunologically (7,8). Other MPs have been described as enzymes or adhesins involved in the pathogenicity of this fungus (10).…”