The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicans is a surface antigen.
A lambda gt11 cDNA library from Candida albicans ATCC 26555 was screened by using pooled sera from two patients with systemic candidiasis and five neutropenic patients with high levels of anti-C. albicans immunoglobulin M antibodies. Seven clones were isolated from 60,000 recombinant phages. The most reactive one contained a 0.9-kb cDNA encoding a polypeptide immunoreactive only with sera from patients with systemic candidiasis. The whole gene was isolated from a genomic library by using the cDNA as a probe. The nucleotide sequence of the coding region showed homology (78 to 79%) to the Saccharomyces cerevisiae TDH1 to TDH3 genes coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and their amino acid sequences showed 76% identity; thus, this gene has been named C. albicans TDH1. A rabbit polyclonal antiserum against the purified cytosolic C. albicans GAPDH (polyclonal antibody [PAb] anti-CA-GAPDH) was used to identify the GAPDH in the beta-mercaptoethanol extracts containing cell wall moieties. Indirect immunofluorescence demonstrated the presence of GAPDH at the C. albicans cell surface, particularly on the blastoconidia. Semiquantitative flow cytometry analysis showed the sensitivity of this GAPDH form to trypsin and its resistance to be removed with 2 M NaCl or 2% sodium dodecyl sulfate. The decrease in fluorescence in the presence of soluble GAPDH indicates the specificity of the labelling. In addition, a dose-dependent GAPDH enzymatic activity was detected in intact blastoconidia and germ tube cells. This activity was reduced by pretreatment of the cells with trypsin, formaldehyde, and PAb anti-CA-GAPDH. These observations indicate that an immunogenic, enzymatically active cell wall-associated form of the glycolytic enzyme GAPDH is found at the cell surface of C. albicans cells.
Gil-Navarro
I
I
Departamento de Microbiologia y Ecologia, Facultad de Farmacia, Universitat de València, Spain.
Gil
M L
ML
Casanova
M
M
O'Connor
J E
JE
Martínez
J P
JP
Gozalbo
D
D
D000908:Antibodies, Fungal / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000946:Antigens, Fungal / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D015139:Blotting, Southern; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D002177:Candidiasis / Q000276:immunology; D002473:Cell Wall; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D005800:Genes, Fungal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D008969:Molecular Sequence Data
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D000954:Antigens, Surface; D018076:DNA, Complementary; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases