Gil-Navarro I, Gil M L, Casanova M, O'Connor J E, Martínez J P, Gozalbo D
Departamento de Microbiologia y Ecologia, Facultad de Farmacia, Universitat de València, Spain.
J Bacteriol. 1997 Aug;179(16):4992-9. doi: 10.1128/jb.179.16.4992-4999.1997.
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.
利用两名系统性念珠菌病患者和五名抗白色念珠菌免疫球蛋白M抗体水平高的中性粒细胞减少患者的混合血清，对来自白色念珠菌ATCC 26555的λgt11 cDNA文库进行筛选。从60,000个重组噬菌体中分离出7个克隆。反应性最强的一个克隆包含一个0.9 kb的cDNA，其编码的多肽仅与系统性念珠菌病患者的血清发生免疫反应。以该cDNA为探针，从基因组文库中分离出整个基因。编码区的核苷酸序列与酿酒酵母中编码甘油醛-3-磷酸脱氢酶（GAPDH）的TDH1至TDH3基因具有同源性（78%至79%），其氨基酸序列具有76%的同一性；因此，该基因被命名为白色念珠菌TDH1。用针对纯化的白色念珠菌胞质GAPDH的兔多克隆抗血清（多克隆抗体[PAb]抗CA-GAPDH）来鉴定含有细胞壁成分的β-巯基乙醇提取物中的GAPDH。间接免疫荧光显示白色念珠菌细胞表面存在GAPDH，尤其是在芽生孢子上。半定量流式细胞术分析表明，这种形式的GAPDH对胰蛋白酶敏感，对2 M NaCl或2%十二烷基硫酸钠具有抗性。可溶性GAPDH存在时荧光的降低表明标记具有特异性。此外，在完整的芽生孢子和芽管细胞中检测到剂量依赖性的GAPDH酶活性。用胰蛋白酶、甲醛和PAb抗CA-GAPDH预处理细胞后，该活性降低。这些观察结果表明，在白色念珠菌细胞表面发现了一种具有免疫原性、酶活性的与细胞壁相关的糖酵解酶GAPDH形式。
