Cloning of cDNAs coding for<i>Candida albicans</i>cell surface proteins

Abstract

Two cDNA libraries were constructed from mRNAs obtained from yeast cells and germ-tubes of Candida albicans in lambda gt11. Immunoscreening with polyclonal antibodies raised against cell wall components allowed the detection of 29 positive clones. Two of these clones were selected for their specific reactivity with antisera either from yeast (clone 11Y) or germ-tubes (clone 24M). cDNA fragments were isolated by the digestion of lambda DNA with EcoRI. Southern blot analysis with these fragments as probes demonstrated homology with C. albicans DNA, and by Northern analysis two mRNAs transcripts were detected with sizes of approximately 1.5 kb for 11Y and 1.1 kb for 24M. Both transcripts were present in yeast cells as well as in germ-tubes. The whole genes were isolated from a C. albicans genomic library in the YRp7 vector by hybridization with the cDNA probes. Monospecific antibodies were purified from polyclonal antisera by affinity for the fusion proteins. Western blot analysis with 11Y-specific antibodies revealed a cross-reactivity with material found in the yeast cell wall as well as in other subcellular fractions, whereas clone 24M codes for a 30 kDa protein detected mainly in the membrane fraction and in the SDS-solubilized material from mycelial cell walls. Sequencing of the cDNA molecules and restriction map of the cloned genes demonstrate that clone 11Y is an enolase previously characterized in C. albicans, whereas clone 24M does not show significant homology with any other cloned gene.